Microparticle and pharmaceutical composition thereof

ABSTRACT

A microparticle includes an agglomerate of a hydrophilic active substance containing particle, which particle includes an amphiphilic polymer composed of a hydrophobic segment of poly (hydroxy acid) and a hydrophilic segment of polysaccharides or polyethylene glycol, and a hydrophilic active substance. It is characterized by an efficient inclusion of the hydrophilic active substance, and a release of the hydrophilic active substance at an appropriate speed in the human body, and is hence very useful as a DDS pharmaceutical preparation.

RELATED APPLICATIONS

This is a §371 of International Application No. PCT/JP2009/052951, withan international filing date of Feb. 20, 2009 (WO 2009/104706 A1,published Aug. 27, 2009), which is based on Japanese Patent ApplicationNos. 2008-041298, filed Feb. 22, 2008, 2008-041299, filed Feb. 22, 2008,2008-167026, filed Jun. 26, 2008, 2008-167027, filed Jun. 26, 2008,2008-243931, filed Sep. 24, 2008, and 2008-243932, filed Sep. 24, 2008,the subject matter of which is incorporated by reference.

TECHNICAL FIELD

This disclosure relates to a microparticle comprising an agglomerate ofparticles containing hydrophilic active substances, and a pharmaceuticalcomposition thereof. Particularly, the disclosure relates to amicroparticle and a pharmaceutical composition thereof as so-called drugdelivery system. More particularly, for example, the disclosure relatesto a microparticle effectively containing a protein, a peptide drugs, anucleic acid drugs, and the like of hydrophilic property and largemolecular weight, and a pharmaceutical composition thereof.

BACKGROUND

Particulate preparations having drugs enclosed in fine particles callednanoparticle, microparticle, nanosphere, microsphere, or microcapsuleare developed, and are attempted to be used as sustained-release agentsfor drugs.

Particulate preparations using polymer compounds as the base includefine particles composed of biodegradable polylactic acid or poly (lacticacid-glycolic acid). In these particulate preparations, it is hard toencapsulate a protein or a peptide drug of hydrophilic property andlarge molecular weight while maintaining the bioactivity. In addition,when administering in the human body, it is known that the drug ismassively released in a short time, and this phenomenon is called aninitial burst.

As fine particles composed of a polymer of covalent bonding ofsaccharide and poly (hydroxy acid), JP 8-19226 discloses a microcapsulefor carrying a pharmacologically active substance composed of a reactionproduct of polyol and polylactic acid. In this technique,polysaccharides are not used, and nothing is mentioned about inclusionof peptide or protein. The microcapsule manufactured by a spray dryingmethod released the encapsulated drug by 62% in 24 hours. This releasespeed is too fast, and the microcapsule can be hardly applied as asustained-release agent for a drug.

JP 2004-521152 and Yuichi Ohya et al., “Encapsulation and/or ReleaseBehavior of Bovine Serum Albumin within and from Polylactide-GraftedDextran Microsphers,” Macromolecular Bioscience, 2004, Vol. 4, pp.458-463 disclose a nanoparticle or a nanoparticle composed of a materialhaving a biodegradable polymer grafted in polysaccharides, but theseliteratures mention nothing about a microparticle composed ofnanoparticles. JP 2004-521152 discloses, for example, a double emulsionmethod already cited in other literatures, as a manufacturing method ofa microparticle for encapsulating a hydrophilic active substance, butthere is no specific description, and inclusion of a drug into aparticle, or release of a drug from a particle is not realized. YuichiOhya et al., “Encapsulation and/or Release Behavior of Bovine SerumAlbumin within and from Polylactide-Grafted Dextran Microsphers,”Macromolecular Bioscience, 2004, Vol. 4, pp. 458-463 discloses amicroparticle encapsulating an albumin manufactured by the doubleemulsion method, but the encapsulation efficiency to the included amountof the albumin is 53% or less, and the low encapsulation efficiency ofthe hydrophilic active substance has a problem in the manufacturingcost.

WO 2006/095668 discloses a fine particle containing an amphiphilicpolymer composed of polysaccharides and an aliphatic polyester, morespecifically a fine particle composed of an inner nucleus ofpolysaccharides, a hydrophobic outer layer of aliphatic polyester, and asurface modifier bonded to the hydrophobic outer layer. This fineparticle does not have an agglomeration structure of fine particles, andspecific examples are not shown about particles of particle diameter ofmicrometer units. The encapsulation efficiency of the hydrophilicsubstance is 50% or less, and this low encapsulation efficiency is asimilar problem as in the case above.

JP 10-511957 discloses a nanoparticle of average particle diameter ofless than 300 nm, composed of a naturally derived polymer of dextran,but specific examples are not shown. This is not an agglomerationstructure of fine particles, and the average particle diameter ishundreds of nanometers, and the drug is likely to diffuse from the siteof administration, and it is not preferred as a sustained-release agent.

As the polymer for forming particles, JP 2004-513154 and JP 2004-514734disclose and suggest use of an amphiphilic block polymer having ahydrophilic portion such as polyethylene glycol, and a hydrophobicportion such as poly(lactic acid-glycolic acid). Micelle particles usingsuch amphiphilic block polymer are usually hydrophobic in the inside,and hydrophilic in the outer layer, and they are suited to containmentof hydrophobic low molecular weight drugs, but not suited to containmentof hydrophilic active substances such as protein or peptide.

JP 2000-501084 and Anshu Yang et al., “Tumor necrosis factor alphablocking peptide loaded PEG-PLGA nanopeptides: Preferparation and invitro evaluation,” International Journal of Pharmaceutics, 2007, Vol.331, pp. 123-132 disclose attempts to contain a protein in a particleusing an amphiphilic block polymer, but the amount of the drug to becontained is small, or the initial burst is large, and so far themanufacturing technology of particles having properties suited assustained-release injection of a hydrophilic drug is not establishedyet.

As mentioned above, microparticles using a polymer have been developed.Hence, it could be helpful to provide a microparticle capable ofencapsulating a hydrophilic active substance efficiently, and moreparticularly a microparticle capable of releasing the encapsulated drugat an appropriate speed, without causing significant initial burst.

SUMMARY

We provide:

-   -   a microparticle including an agglomerate of hydrophilic active        substance containing particles, which particle includes an        amphiphilic polymer composed of a hydrophobic segment of poly        (hydroxy acid) and a hydrophilic segment of polysaccharides or        polyethylene glycol, and a hydrophilic active substance, or more        particularly a microparticle comprising agglomerate of        hydrophilic active substance containing particles, which        particle has a hydrophilic segment of an amphiphilic polymer in        the inside and has an outer layer of the hydrophobic segment of        the amphiphilic polymer;    -   a method for manufacturing a microparticle including forming a        reversed-phase emulsion by mixing an aqueous solvent containing        a hydrophilic active substance and a water-immiscible organic        solvent dissolving an amphiphilic polymer, obtaining a solid        content containing a hydrophilic active substance by removing        the solvent from the reversed-phase emulsion, and introducing        the solid content or a dispersion liquid containing the solid        content into a liquid phase containing a surface modifier; and    -   a pharmaceutical preparation including the microparticles.

The microparticle is capable of encapsulating a hydrophilic activesubstance efficiently, and releasing the hydrophilic active substance atan appropriate speed in the human body, and is hence usable as a novelDDS preparation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows drug release from microparticles encapsulating human growthhormone.

FIG. 2 shows drug release from dextran-PLGA microparticles encapsulatinghuman insulin.

FIG. 3 shows an SEM image of dextran-PLGA microparticles.

FIG. 4 shows an SEM image of polyethyleneglycol-poly(epsilon-caprolactone) microparticle.

FIG. 5 shows time-course changes of blood drug concentration in mouseadministered human growth hormone-encapsulating particlessubcutaneously.

FIG. 6 shows time-course changes of blood drug concentration in mouseadministered human growth hormone-encapsulating microparticlessubcutaneously.

FIG. 7 shows changes of body weight in mouse administered human growthhormone-encapsulating microparticles subcutaneously.

FIG. 8 shows time-course changes of blood IGF-1 concentration in mouseadministered human growth hormone-encapsulating microparticlessubcutaneously.

FIG. 9 shows drug release in a buffer solution ofExendin-4-encapsulating microparticles.

FIG. 10 shows time-course changes of blood drug concentration in mouseadministered Exendin-4-encapsulating microparticles subcutaneously.

FIG. 11 shows drug release from human growth hormone-encapsulatingassociated particles microparticles.

FIG. 12 shows the relation between particle diameter and amount ofdimethyl carbonate added at the time of preparation of S/O/W typeemulsion.

FIG. 13 shows results of enclosure rate entrappement efficiency of FD40encapsulating microparticles.

FIG. 14 shows an SEM image of microparticle powder prepared fromPEG-PLGA polymer (5k-10k).

FIG. 15 shows an SEM image of microparticle powder prepared fromPEG-PLGA polymer (5k-61k).

FIG. 16 shows release behavior of FD40 from FD40-encapsulatingmicroparticles.

FIG. 17 shows release behavior of drug from human insulin-encapsulatingmicroparticles.

FIG. 18 shows time-course changes of blood drug concentration in mouseadministered human growth hormone-encapsulating microparticlessubcutaneously.

FIG. 19 shows time-course changes of blood drug concentration in mouseadministered human growth hormone-encapsulating microparticlessubcutaneously.

FIG. 20 shows time-course changes of blood IGF-1 concentration in mouseadministered human growth hormone-encapsulating microparticlessubcutaneously.

FIG. 21 shows time-course changes of blood pharmacokinetics in mouseadministered Exendin-4-encapsulating microparticles subcutaneously.

FIG. 22 shows the relation between particle diameter and amount ofdimethyl carbonate added at the time of preparation of S/O/W typeemulsion.

DETAILED DESCRIPTION

We form a microparticle by agglomeration of hydrophilic active substancecontaining particles which particle comprises an amphiphilic polymer anda hydrophilic active substance. The agglomeration is bonding of two ormore particles by way of inter-particle force or other substance, andforming of a set. The inter-particle force is not specifiedparticularly, but usable examples include hydrophobic interaction,hydrogen bond, and van der Waals force. The agglomeration is not limitedto a state of mutual contact of particles, but substances having anaffinity for particles may be present among particles, or particles maydistribute in a matrix. As the substances having affinity for particlesor the matrix, a polymer is preferred. By agglomeration of thehydrophilic active substance containing particles, as compared with asingle particle, the effect that the encapsulation efficiency of thehydrophilic active substance is higher is attained. The particlediameter of the hydrophilic active substance containing particles to beassociated is variable.

Microparticles are particles having the particle diameter ranging fromsub-microns to sub-millimeters. The average particle diameter ofmicroparticles is not particularly limited, but in the case ofadministration of the microparticles by injection to the human body, thegreater the average particle diameter, the larger is the syringe needle,and the patient's burden is increased, and therefore from the viewpointof lowering of the patient's burden, it is preferred to be in a range of1 μm to 50 μm. The average particle diameter of microparticles may bedetermined by image analysis by using a scanning electron microscope.

The number of agglomerations of hydrophilic active substance containingparticles for composing a microparticle is preferred to be in a rangefrom 10 to the seventh power of 10, more preferably in a range from thefifth power of 10 to the seventh power of 10. The number ofagglomerations is calculated from the average particle diameter ofhydrophilic active substance containing particles and the averageparticle diameter of microparticles.

The amphiphilic polymer is composed of a hydrophobic segment ofpoly(hydroxy acid) and a hydrophilic segment of polysaccharides orpolyethylene glycol. The amphiphilic property is a state having bothhydrophilic and hydrophobic properties, and as for the hydrophilicproperty, when solubility in water is higher in a certain segment thanin other segments, such segment is said to be hydrophilic. A hydrophilicsegment is preferred to be soluble in water, but if hardly soluble, itis hydrophilic if solubility in water is higher than other segments. Acertain segment is said to be hydrophobic if solubility in water islower than other parts. A hydrophobic segment is preferred to beinsoluble in water, but if soluble, it can be hydrophobic if solubilityin water is lower than other segments.

Specific examples of poly(hydroxy acid) of the amphiphilic polymerinclude polyglycolic acid, polylactic acid, poly(2-hydroxy butyricacid), poly(2-hydroxy valeric acid), poly(2-hydroxy caproic acid),poly(2-hydroxy capric acid), poly(malic acid), and derivatives andcopolymers of these high molecular compounds. However, sincemicroparticles are desired to have no significant effects at the time ofadministration in human body, the poly(hydroxy acid) of amphiphilicpolymer is also preferred to be a biocompatible high polymer. Thebiocompatible high polymer is a substance not having significant effectson the human body when administered, and more specifically the LD50 ispreferred to be 2,000 mg/kg or more by oral administration of the highpolymer in rat.

As poly(hydroxy acid) of the biocompatible high polymer, a copolymer ofpolylactic acid, and polyglycolic acid, or poly(lactic acid-glycolicacid) is preferred. When the poly(hydroxy acid) is a poly(lacticacid-glycolic acid), the composition ratio of the poly(lacticacid-glycolic acid) (lactic acid/glycolic acid) (mol/mol %) is notparticularly limited, but the ratio is preferably 10/0 to 30/70, or morepreferably 60/40 to 40/60.

When the hydrophilic segment of the amphiphilic polymer ispolysaccharides, examples of the polysaccharides may include cellulose,chitin, chitosan, gellan gum, alginic acid, hyaluronic acid, pullulan,or dextran, and dextran is most preferable.

The amphiphilic polymer is preferably composed by graft polymerizationof graft chain(s) of poly(hydroxy acid) in a main chain ofpolysaccharide. The average molecular weight of the main chain ofpolysaccharide is preferably 1,000 to 100,000, or more preferably 2,000to 50,000, and the average molecular weight of the poly(hydroxy acid) ispreferably 500 to 100,000, or more preferably 1,000 to 10,000. The valueof average molecular weight of poly(hydroxy acid) to the averagemolecular weight of polysaccharides is preferably 0.01 times to 100times, more preferably 0.02 times to 10 times, or most preferably 0.02times to 1 times.

The number of graft chains of poly(hydroxy acid) bonded with the mainchain of polysaccharides is preferably 2 to 50. The number of graftchains may be determined from the average molecular weight of graft typeamphiphilic polymer, main chain of polysaccharides, and graft chain ofpoly(hydroxy acid).

When the hydrophilic segment of the amphiphilic polymer is polyethyleneglycol, the amphiphilic polymer is preferred to be a block polymer ofpolyethylene glycol and poly(hydroxy acid). The term “block” refers to aportion segment of a polymer molecule, consisting of at least five ormore monomer units, and being different in chemical structure orconfiguration between a portion segment and other adjacent portionsegment, and a polymer formed of two or more blocks coupled straightlyis called a block polymer. Each block forming a block polymer maycomprise two or more monomer units, that is, a random, alternating, orgradient polymer may be formed. When the hydrophilic segment of theamphiphilic polymer is polyethylene glycol, the amphiphilic polymer ispreferred to be a block polymer coupling one each of polyethylene glycoland polyhydroxy acid.

When the hydrophilic segment of the amphiphilic polymer is polyethyleneglycol, specific examples of the polyethylene glycol to be used includestraight-chain or branched polyethylene glycol or its derivatives, and apreferred example of polyethylene glycol derivative is polyethyleneglycol monoalkyl ether. The alkyl group of the polyethylene glycolmonoalkyl ether is a straight-chain or branched alkyl group having 1 to10 carbon atom(s), and a branched alkyl group having 1 to 4 carbonatom(s) is more preferable, and methyl, ethyl, propyl, and iso-propylgroups are particularly desired.

The average molecular weight of the polyethylene glycol is notparticularly limited, but is preferably 2,000 to 15,000, more preferably2,000 to 12,000, even more preferably 4,000 to 12,000, and particularlypreferably 5,000 to 12,000.

When the hydrophilic segment of the amphiphilic polymer is polyethyleneglycol, the average molecular weight of the poly(hydroxy acid) is notparticularly limited, but is preferably 5,000 to 200,000, morepreferably 15,000 to 150,000, or even more preferably 20,000 to 100,000.The value of average molecular weight of poly(hydroxy acid) to theaverage molecular weight of polyethylene glycol is preferably 1.0 timesor more, more preferably 2 times or more, most preferably 4 times ormore, and particularly preferably 4 times or more to 25 times or less.

The average molecular weight refers to the number-average molecularweight unless otherwise specified, and the number-average molecularweight is an average molecular weight calculated by a method notconsidering weighting of magnitude of a molecule, and the averagemolecular weight of amphiphilic polymer, polysaccharides, andpolyethylene glycol can be obtained as the molecular weight convertedinto polystyrene or pullulan measured by gel permeation chromatography(GPC). The average molecular weight of poly(hydroxy acid) can bedetermined from the ratio of peak integral value of terminal residue andpeak integral value of others than terminal residue as measured bynuclear magnetic resonance (¹H-NMR) measurement.

The amphiphilic polymer composed of polysaccharides and poly(hydroxyacid) may be synthesized by any known method and, as far as areversed-phase emulsion can be formed, the synthesizing method is notspecified, and it can be manufactured, for example, in any one of thefollowing methods (1), (2), and (3):

-   -   1) In the presence of a tin catalyst, a hydroxy acid activating        monomer is added to polysaccharides to carry out a        polymerization reaction, and poly (hydroxy acid) is further        added, and a graft type amphiphilic polymer is manufactured        (Macromolecules, 31, 1032-1039 (1998)).    -   2) The hydroxyl group of partially non-protected polysaccharides        of which majority of hydroxy group is protected by a substituent        is activated by a base, a hydroxy acid activating monomer is        added to form graft chain(s) composed of poly(hydroxy acid), and        finally the protective group is removed, and a graft type        amphiphilic polymer is manufactured (Polymer, 44, 3927-3933,        (2003)).    -   3) In polysaccharides, a copolymer of poly(hydroxy acid) is        added to execute condensation reaction by using a dehydrating        agent and/or a functional activating agent, and a graft type        amphiphilic polymer is manufactured (Macromolecules, 33,        3680-3685 (2000)).

The amphiphilic polymer composed of polyethylene glycol and poly(hydroxyacid) may be synthesized by any known method and, as far as areversed-phase emulsion can be formed, the synthesizing method is notspecified and, for example, in the presence of a tin catalyst, a hydroxyacid activating monomer is added to polyethylene glycol to carry out apolymerization reaction to form poly(hydroxy acid), and an amphiphilicblock polymer is manufactured (Journal of Controlled Release, 71,203-211 (2001)).

The structure of the hydrophilic active substance containing particlecomprising the amphiphilic polymer and hydrophilic bioactive substanceis not particularly limited, but as far as the hydrophilic activesubstance containing particle has a hydrophilic segment of anamphiphilic polymer in the inside, and has an outer layer of ahydrophobic segment of an amphiphilic polymer, it is preferable becausethe contained hydrophilic active substance can be maintained morestably.

When the hydrophilic active substance containing particle is a particlehaving a hydrophilic segment of an amphiphilic polymer in the inside,and having an outer layer of a hydrophobic segment of an amphiphilicpolymer, it is one of the preferred embodiments if a surface modifier isbonded to the outer layer of poly(hydroxy acid). Bonding may be eithernon-covalent bonding or covalent bonding. Non-covalent bonding ispreferably hydrophobic interaction, but may include electrostaticinteraction, hydrogen bond, or van der Waals force, or a combinationthereof. In non-covalent bonding, the hydrophobic outer layer of fineparticles containing the amphiphilic polymer, and the hydrophobicportion of a surface modifier described below may be preferably bondedto each other by hydrophobic interaction. In this case, the dispersantof fine particles is particularly preferred to be water, buffersolution, physiological saline, surface modifier aqueous solution, orfine particle dispersant of hydrophilic solvent.

The surface modifier is preferably a compound capable of stabilizing thewater-oil interface of S/O/W type emulsion, or the oil-oil emulsioninterface of S/O1/O2 type emulsion, and more preferably a compoundhaving properties for enhancing the colloid stability of microparticles.The surface modifier may be one type or a mixture of plural types. Theproperty of enhancing the colloid stability means to prevent or delayaggregation of microparticles in the solvent.

The surface modifier is preferred to be an amphiphilic compound or ahydrophilic polymer.

The hydrophilic polymer of the surface modifier is preferably any oneselected from the group consisting of polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, polyethylene imine, polyacrylic acid,polymethacrylic acid, poly-1,3-dioxolane, 2-methacryloyl oxyethylphosphoryl choline polymer, poly-1,3,6-trioxane, polyamino acid,peptide, protein, saccharides, and analogs thereof.

Analogs of the hydrophilic polymer may include a surfactant having ahydrophilic polymer partially modified by a hydrophobic group such as along-chain alkyl, but are not particularly limited to this.

As a polyethylene glycol analog of a surface modifier, it is preferredto use Pluronic (registered trademark) commercially available by BASF orits equivalents.

As a polyamino acid of a surface modifier, polyaspartic acid,polyglutamic acid, or their analogs may be preferably used. An analogintroducing a long-chain alkyl group in part of polyaspartic acid orpolyglutamic acid is particularly preferable.

As a peptide of a surface modifier, a basic peptide may be used.

As a protein of a surface modifier, gelatin, casein, or albumin ispreferred for enhancing the dispersion performance of particles. As aprotein, an antibody is one of preferred examples.

As saccharides of a surface modifier, monosaccharides, oligosaccharides,and polysaccharides are preferable. As polysaccharides, cellulose,chitin, chitosan, gellan gum, alginic acid, hyaluronic acid, pullulan,and dextran are preferable. Particularly, cholesteryl pullulan ispreferable in view of better dispersibility of particles. Analogs of anyone selected from the group consisting of cellulose, chitin, chitosan,gellan gum, alginic acid, hyaluronic acid, pullulan, and dextran arepreferable.

As the surface modifier, these examples of peptide, protein, andsaccharides are particularly preferred to be analogs partly modifyingthe hydrophobic group of a long-chain alkyl, or analogs modifying thehydrophilic polymer or the amphiphilic compound.

In the surface modifier, the amphiphilic compound includes a lipid asone of the preferred examples.

In the surface modifier, the amphiphilic compound includes a surfactantas one of the preferred examples. Preferred examples of the surfactantinclude: nonionic active agents such as polyoxyethylene-polypropyleneglycol copolymer, sucrose fatty acid ester, polyethylene glycol fattyacid ester, polyoxyethylene sorbitan mono-fatty acid ester,polyoxyethylene sorbitan di-fatty acid ester, polyoxyethylene glycerinmono-fatty acid ester, polyoxyethylene glycerin di-fatty acid ester,polyglycerin fatty acid, polyoxyethylene castor oil, polyoxyethylenehardened castor oil; alkyl sulfates such as lauryl sodium sulfate,lauryl ammonium sulfate, stearyl sodium sulfate; or lecithin.

The hydrophilic active substance is exemplified by low molecularcompound, protein, peptide, DNA, RNA, or modifying nucleic acid. Even ahydrophobic drugs may be contained in the microparticle if madehydrophilic by using a solubilizing agent. The solubilizing agent hereinpreferably includes cyclodextrin and its analogs.

The protein or the peptide used as the hydrophilic active substance isnot particularly limited, but a bioactive protein or a bioactive peptideis preferred. The bioactive protein or the bioactive peptide includepeptide hormone, cytokine, enzyme protein, or antibody. Specificexamples include: GLP-1 receptor antagonist peptide such as Exendin-4,parathyroid hormone (PTH), calcitonin, insulin, insulin-like growthfactor, angiotensin, glucagon, GLP-1; bombesin, motilin, gastrin, growthhormone, prolactin (luteotropic hormone), gonadotropin (gonadotropichormone), thyrotropic hormone, adrenocorticotropic hormone (ACTH), ACTHderivative (ebiratide), melanocyte stimulating hormone, folliclestimulating hormone (FSH), sermorelin, vasopressin, oxytocin,protirelin, leuteinizing hormone (LH), corticotropin, secretin,somatropin, thyrotropin (thyroid stimulating hormone), stomatostatin,gonadotropin releasing hormone (GnRH), G-CSF, erythropoetin (EPO),thrombopoetin (TPO), megakaryocyte potentiator, HGF, EGF, VEGF,interferon α, interferon β, interferon γ, interleukins, FGF (fibroblastgrowth factor), BMP (bone marrow proteins), thymic humor factor (THF),serum thymic factor (FTS), superoxide dimustase (SOD), urokinase,lisozyme, tissue plasminogen activator, asparakinase, kallikrein,Ghrelin, adiponectin, leptin, atrial sodium diuretic peptide, atrialsodium diuretic factor, cerebral sodium diuretic peptide (BNP),conantokin G, dynorphin, endorphin, Kyotorphin, enkephalin, neurotensin,angiostin, bradykinin, substance P, kalidin, hemoglobin, protein C, VIIafactor, glycocerebrosidase, streptokinase, staphylokinase, thymosin,pancreozimine, cholecistokinin, human placenta lactogen, tumor necrosisfactor (TNF), polymixin B, cholistine, gramicidin, bacitracin,thymopoetin, bombecin, cerulein, thymostimulin, secretin, resistin,hepcidin, neuropetide Y, neuropeptide S, cholecistokinine-pancreozimine(CCK-PZ), brain-derived nutrient factor (BDNF), vaccine, and the like.These bioactive proteins or bioactive peptides may be natural proteinsor peptides, or derivatives modified in part of their sequence, orcompounds modified by polyethylene glycol or sugar chain.

When the hydrophilic active substance is DNA, RNA, or modifying nucleicacid, it may be any one of cationic surfactant, cationic lipid, cationicpolymer, or other compounds complexed with the analogs thereof.

Saccharides used as the hydrophilic active substance include hyaluronicacid, heparin, dextran sulfate, dextran or FITC labeled dextran (forexample, FD40, etc.).

We also provide a method for manufacturing the microparticle formed byagglomeration of the hydrophilic active substance containing particles,the method comprising:

-   -   a) a step of forming a reversed-phase emulsion by mixing an        aqueous solvent containing the hydrophilic active substance and        a water-immiscible organic solvent dissolving the amphiphilic        polymer,    -   b) a step of obtaining a solid content containing the        hydrophilic active substance by removing the solvent from the        reversed-phase emulsion, and    -   c) a step of introducing the solid content or a dispersion        liquid containing the solid content into a liquid phase        containing the surface modifier.

In the method for manufacturing the microparticle formed byagglomeration of the hydrophilic active substance containing particles,the reversed-phase emulsion is formed by adding an aqueous solventcontaining the hydrophilic active substance to a water-immiscibleorganic solvent dissolving an amphiphilic polymer and mixing them. Ifnecessary, it is possible to use, for example, an agitating device suchas magnetic stirrer, a turbine agitating device, a homogenizer, or amembrane emulsifying device provided with a porous film. Thewater-immiscible organic solvent is an organic solvent of whichsolubility in water is 30 g (water-immiscible organic solvent)/100 ml(water) or less, and other organic solvents of which solubility in wateris higher than the specified value are characterized as water-miscibleorganic solvents.

As the aqueous solution at step (a), water or water solution containinga water-soluble substance is used. The water-soluble substance may beany one of inorganic salts, saccharides, organic salts, amino acid, andthe like.

The property of water immiscible organic solvent at step (a) is notparticularly limited, but it is preferably a solvent capable ofdissolving poly(hydroxyl acid) as the hydrophobic segment of theamphiphilic polymer, and hardly dissolving or not dissolving thehydrophilic segment. The water-immiscible organic solvent is preferredto be dissipated and removed by freeze-drying or the like, and ispreferred to be 0.1 g (water-immiscible organic solvent)/100 ml (water)or less. Specific examples of the water-immiscible organic solventinclude ethyl acetate, isopropyl acetate, butyl acetate, dimethylcarbonate, diethyl carbonate, methylene chloride, and chloroform. Theratio of the water-immiscible organic solvent to the aqueous solvent ispreferably 1,000:1 to 1:1, more preferably 100:3 to 3:1. Theconcentration of the amphiphilic polymer in the water-immiscible organicsolvent varies with the type of the water immiscible organic solvent orthe amphiphilic polymer, but is preferably 0.01 to 90% (w/w), morepreferably 0.1 to 50% (w/w), or even more preferably 1 to 20% (w/w).

At step (a), in the process of forming a reversed-phase emulsion by theaqueous solvent containing the hydrophilic active substance and thewater-immiscible organic solvent dissolving the amphiphilic polymer,depending on the pharmacological purpose, a reversed-phase emulsion maybe formed by using a water-immiscible organic solvent dissolving two ormore types of amphiphilic polymer.

At step (a), in the process of forming a reversed-phase emulsion by theaqueous solvent containing the hydrophilic active substance and thewater-immiscible organic solvent dissolving the amphiphilic polymer, toassist formation of the reversed-phase emulsion and to form a uniformand fine reversed-phase emulsion, an assisting agent may be added. Suchassisting agent may be preferably a compound selected from the groupconsisting of alkyl alcohol having 3 to 6 carbon atoms, alkyl aminehaving 3 to 6 carbon atoms, and alkyl carboxylic acid having 3 to 6carbon atoms. The structure of alkyl chain of these assisting agents isnot specified particularly, and either straight-chain structure orbranched structure may be applicable, or saturated alkyl ornon-saturated alkyl may be usable. In particular, tert-butanol,iso-butanol, and pentanol are preferred as the assisting agent.

The average particle diameter of the reversed-phase emulsion at step (a)is variable with the particle diameter of the desired microparticle, andis not particularly limited, but to manufacture a microparticle for apharmaceutical preparation which is one of the applications of themicroparticle, the upper limit of the average particle diameter ispreferably 50 μm, more preferably 5 μm, even more preferably 500 nm,particularly preferably 150 nm, and most preferably 100 nm. The lowerlimit of the average particle diameter of the reversed-phase emulsion ispreferably 10 nm, or more preferably 50 nm.

Next, in the manufacturing method of a microparticle, it is important toinclude step (b) of obtaining a solid content containing the hydrophilicactive substance by removing the solvent from the reversed-phaseemulsion obtained at step (a).

At step (b), the method of removing the solvent from the reversed-phaseemulsion is not particularly limited, but may include, for example,heating, in-vacuo drying, dialysis, freeze-drying, centrifugaloperation, filtration, re-sedimentation, and a combination thereof.

Among these methods of removing the solvent from the reversed-phaseemulsion, freeze-drying is particularly preferred because it is small instructural changes due to uniting of particles in the reversed-phaseemulsion, and is capable of avoiding degeneration due to hightemperature of the hydrophilic active substance. The condition and theapparatus of freeze-drying include a freezing process and a dryingprocess at reduced pressure, and the process is particularly preferredto consist of preliminary freezing step as an ordinary method offreeze-drying, a primary drying step at reduced pressure and lowtemperature, and a secondary drying step at reduced pressure. Forexample, by cooling and freezing below the melting point of aqueoussolution and water immiscible organic solvent, for composing areversed-phase emulsion, and then drying at reduced pressure, afreeze-dried hydrophilic active substance containing solid content isobtained. The temperature of preliminary freezing may be determinedproperly by experiment considering from the solvent composition, and isgenerally preferred to be −20° C. or less. The degree of reducedpressure in the drying process may be determined properly by experimentconsidering from the solvent composition, and is generally preferred tobe 3,000 Pa or less, or more preferably 500 Pa or less, for shorteningof the drying time. For freeze-drying, it is preferred to employ afreeze-drying apparatus for laboratory having a cold trap andconnectable to a vacuum pump, or a rack type vacuum freeze-dryingapparatus used in manufacture of pharmaceutical preparations, and afterpreliminary freezing by using liquid nitrogen or refrigerant, drying atreduced pressure is executed at cooled temperature or room temperatureby using a vacuum pump or other pressure reducing device.

The solid content containing the hydrophilic active substance obtainedat step (b) is obtained as an aggregate of hydrophilic active substancecontaining particles comprising the amphiphilic polymer, which aggregateconforms to the structure of the reversed-phase emulsion. The aggregateis an irregular mass gathering fine particles by inter-particle force,and is clearly distinguished in shape from the microparticle. Theaverage particle diameter of the hydrophilic active substance containingfine particles for forming this aggregate is variable with the particlediameter of the desired microparticle, and is not particularly limited,but to manufacture a microparticle for a pharmaceutical preparationwhich is one of the applications of the microparticle, the upper limitof the average particle diameter is preferably 50 μm, more preferably 5μm, most preferably 500 nm, especially 150 nm, particularly 100 nm. Thelower limit of the average particle diameter of the hydrophilic activesubstance containing fine particles is preferably 10 nm, or morepreferably 50 nm.

In the method for manufacturing the microparticle, it is important toinclude step (c) of introducing the solid content containing thehydrophilic active substance or a dispersion liquid containing the solidcontent in a liquid phase containing the surface modifier.

At step (c), the method of introducing the solid content or thedispersion liquid containing the solid content in a liquid phasecontaining the surface modifier includes, for example, a method ofadding the solid content in a liquid phase containing the surfacemodifier, and a method of dispersing the solid content once in adispersion medium, and adding the obtained dispersion liquid(solid-in-oil (S/O) suspension) in a liquid phase containing the surfacemodifier.

When dispersing the solid content containing the hydrophilic activesubstance once in a dispersion medium, the dispersion medium is notparticularly limited, but is preferably a solvent capable of dissolvingpoly(hydroxy acid), but not dissolving substantially the hydrophilicsegment composing the amphiphilic polymer, for the purpose of sustainingthe hydrophilic active substance containing particle structure composedof the amphiphilic polymer having the structure of the reversed-phaseemulsion for composing the hydrophilic active substance containing solidcontent. The solvent capable of dissolving poly(hydroxy acid), but notdissolving substantially the hydrophilic segment is a solvent of whichsolubility of hydrophilic segment in the solvent is 50 mg/mL or less,preferably 10 mg/mL or less.

The dispersion medium may be either water-immiscible organic solvent orwater miscible organic solvent as far as having the features mentionedabove, and the water-immiscible organic solvent is more preferable.Specific examples of the water-immiscible organic solvent capable ofdissolving poly(hydroxy acid) of amphiphilic polymer, but not dissolvingsubstantially in the hydrophilic segment include ethyl acetate,isopropyl acetate, butyl acetate, dimethyl carbonate, diethyl carbonate,methylene chloride, chloroform, dioxane, toluene, and xylene.

The dispersion medium for dispersing the solid content containing thehydrophilic active substance may contain various additives soluble inthe dispersion medium, for the purpose of controlling the releasingspeed of the hydrophilic active substance due to decomposition ordisintegration of the hydrophilic active substance containing particles,for example, an acidic compound, a basic compound, an amphiphilicpolymer, or a biodegradable polymer.

The liquid phase at step (c) is preferably capable of dissolving thesurface modifier, and is higher in boiling point than the hydrophilicactive substance containing solid content dispersion medium, and mayinclude any one of aqueous solvent, water-immiscible organic solvent,and water miscible organic solvent. The aqueous solvent is water, orwater solution containing a water soluble component, and the watersoluble component includes, or example, inorganic salts, saccharides,organic salts, and amino acids; the water-immiscible organic solventincludes, for example, silicone oil, sesame oil, soybean oil, corn oil,cotton seed oil, coconut oil, linseed oil, mineral oil, castor oil,hardened castor oil, liquid paraffin, n-hexane, n-heptane, glycerol, andoleic oil; and the water miscible organic solvent includes, for example,glycerin, acetone, ethanol, acetic acid, dipropylene glycol, triethanolamine, and triethylene glycol. The liquid phase at step (c) ispreferably an aqueous solvent or a water miscible organic solvent. Whenthe liquid phase is an aqueous solvent, and the dispersion medium is awater-immiscible organic solvent, the suspension obtained at step (c) isa so-called solid-in-oil-water (S/O/W) type emulsion, and when theliquid phase is water-immiscible organic solvent or water miscibleorganic solvent, and is not miscible in the dispersion medium, it is asolid-in-oil-in-oil (S/O1/O2) type emulsion.

The ratio by volume of the liquid phase to the dispersion medium fordispersing the hydrophilic active substance containing solid content isgenerally 1,000:1 to 1:1,000, or preferably 100:1 to 1:100.

The concentration of the surface modifier in the liquid phase isvariable with the type of the surface modifier, and is preferably 0.01to 90% (w/v), more preferably 0.1 to 50% (w/v), or even more preferably5 to 10% (w/v).

The surface modifier may be bonded to a poly(hydroxy acid) outer layerof the amphiphilic polymer of the microparticle, and the bonding amountin this case is preferably 0.0001% to 1% of the weight of themicroparticle.

In the liquid phase at step (c), in addition to the surface modifier,various additives may be added depending on the pharmacological purpose,such as buffer agent, antioxidant, salt, polymer, or sugar.

At step (c), it is also preferred to add inorganic salts in the liquidphase. Inorganic salts are preferred to be alkaline metal salt oralkaline earth metal salt, and sodium chloride is particularlypreferable. The concentration of inorganic salts in the liquid phase ispreferably 0 to 1 M, more preferably 10 mM to 1 M, or even morepreferably 10 mM to 100 mM.

At step (c), to manufacture a microparticle of a smaller particle size,the formed solid-in-oil-in-water (S/O/W) type emulsion orsolid-in-oil-in-oil (S/O1/O2) type emulsion may be processed by anemulsifying operation. The emulsifying method is not particularlylimited as far as a stable emulsion can be manufactured. For example,the method includes an agitating method, or a method by using ahigh-pressure homogenizer, or a high-speed homo-mixer.

At step (c), when the dispersion liquid obtained by dispersing the solidcontent containing the hydrophilic active substance once in thedispersion medium is added in the liquid phase containing the surfacemodifier, by removing the dispersion medium, a desired suspension of themicroparticle formed by agglomeration of the hydrophilic activesubstance containing particles is obtained. The method of removing thedispersion medium is not particularly limited, but may include methodsof drying in liquid, dialysis, freeze-drying, centrifugal operation,filtration, and re-sedimentation, and drying in liquid or freeze-dryingmay be particularly preferred. At step (c), when an aqueous solvent isused as the liquid phase, an aqueous dispersant of the microparticle isobtained in this process.

By removing the liquid phase from the microparticle dispersant obtainedin this process, the microparticle can be obtained. The method ofremoving the liquid phase is not particularly limited, but maypreferably include methods of distilling-away by evaporation, dialysis,freeze-drying, centrifugal operation, and filtration.

Fields of application of the microparticle are wide, and versatile, andit is particularly used as a pharmaceutical preparation. When themicroparticle is used as the pharmaceutical preparation, aside frommicroparticles, various pharmacological useful additives may becontained, and usable additives include buffer agent, antioxidant, salt,polymer, or sugar.

When the microparticle is used as a pharmaceutical preparation, themethod of administration includes, for example, oral administration andparental administration, and the parental administration is preferred.The parental administration includes hypodermic administration,intramuscular administration, enteric administration, pulmonaryadministration, local administration (nose, skin, eye), and body cavityadministration, and the hypodermic and intramuscular injections arepreferred in particular. The dose and the number of times ofadministration of the pharmaceutical preparation in the body of thepatient may be properly selected depending on the hydrophilic activesubstance, route of administration, age and body weight of the patient,or severity of the symptom, but usually a dose of 0.1 μg to 100 mg,preferably 1 μg to 10 mg is administered per day per adult person.

Examples

Examples are shown below, but this disclosure is not limited to theexamples described herein.

Example 1 Synthesis of dextran-polylactic Acid (PLA) 1.1 Synthesis ofTMS-dextran (compound 1)

Dextran (NACALAI TESQUE, INC. NAKARAI standard special grade conformingproduct, number-average molecular weight: 13000, 5.0 g) was added toformamide (100 ml), and heated to 80° C. In this solution,1,1,1,3,3,3-hexamethyldisilazane (100 ml) was added by dropping for 20minutes. After dropping, the solution was stirred for 2 hours at 80° C.After completion of the reaction, the reaction solution was returned toroom temperature, and the solution was separated into two layers by adispensing funnel. The upper layer was concentrated at reduced pressure,and methanol (300 ml) was added, and the obtained solid content wasfiltered and dried, and TMS-dextran (11.4 g) was obtained as white solidcontent.

1.2 Synthesis of TMS-dextran-PLA (compound 2)

Compound 1 (0.5 g) and tert-butoxy potassium (35 mg) were dried for 1hour at reduced pressure, and tetrahydrofurane (20 ml) was added, andthe mixture was stirred for 1 hour at room temperature. In thissolution, tetrahydrofurane (20 ml) solution of (L)-lactide (4.49 g) wasdropped, and the mixture was stirred for 5 minutes. After completion ofreaction, the solvent was concentrated at reduced pressure, and purifiedby reprecipitation by a chloroform-methanol system, and TMS-dextran-PLA(1.9 g) was obtained as white solid content.

1.3 Synthesis of dextran-PLA (compound 3)

In chloroform (24 ml) solution of compound 2 (1.9 g), methanol (10.8 ml)and 12N hydrochloric acid (1.2 ml) were added, and stirred for 30minutes at room temperature. The solvent was distilled away at reducedpressure, and the residue was dissolved in chloroform (10 ml), anddropped into diethyl ether cooled to 0° C., and the product wasdeposited. The deposition matter was filtered away, and concentrated atreduced pressure, and dextran-PLA (1.6 g) was obtained. Theweight-average molecular weight of this polymer was 48720, and thenumber-average molecular weight was 43530. (Measurement by GPC: columnToso TSK-gel α-5000×2, DMF system solvent, detector RI, standardproduct, pullulan). The average molecular weight of the graft chain ofthis polymer determined by ¹H-NMR measurement was 2300. The number ofgraft chains was 10 to 12.

Example 2 Synthesis of dextran-poly(lactic acid-glycolic acid) (PLGA)2.1 Synthesis of TMS-dextran-PLGA (compound 4, compound 5, compound 6)

Compound 1 (0.5 g) and tert-butoxy potassium (35 mg) were dried for 1hour at reduced pressure, and tetrahydrofurane (10 ml) was added, andthe mixture was stirred for 1 hour at room temperature. In thissolution, tetrahydrofurane (15 ml) solution of (DL)-lactide (1.12 g) andglycolide (0.9 g) was dropped, and the mixture was stirred for 5minutes. After completion of reaction, the solvent was concentrated atreduced pressure, and purified by reprecipitation by achloroform-methanol system, and TMS-dextran-PLGA (1.96 g) was obtainedas white solid content (compound 4). In the same manner, by the chargingamount of (DL)-lactide (0.784 g) and glycolide (0.63 g), compound 5 wassynthesized, and by the charging amount of (DL)-lactide (1.12 g) andglycolide (0.9 g), compound 6 was synthesized.

2.2 Synthesis of dextran-PLGA (compound 7, compound 8, compound 9)

In chloroform (14 ml) solution of compound 4 (1.96 g), methanol (6.3 ml)and 12N hydrochloric acid (0.7 ml) were added, and stirred for 30minutes at room temperature. The solvent was distilled away at reducedpressure, and the residue was dissolved in chloroform (10 ml), anddropped into diethyl ether cooled to 0° C., and the product wasdeposited. The deposition matter was filtered away, and concentrated atreduced pressure, and dextran-PLGA (1.25 g) was obtained (compound 7).From compounds 5 and 6, dextran-PLGA products were obtained as the samemanner except that trifluoroacetic acid was used (compound 8, compound9). The weight-average molecular weight and the number-average molecularweight of the polymer of compounds 7 to 9 were determined by GPCmeasurement (column Toso TSK-gel α-5000×2, DMF system solvent, detectorRI, standard product, pullulan). The average molecular weight of thegraft chain and the number of graft chains were determined by ¹H-NMRmeasurement.

As for compound 7, the weight-average molecular weight was 43,820, thenumber-average molecular weight was 33,422, the graft chain molecularweight was 1,900, and the number of graft chains was 7 to 10.

As for compound 8, the weight-average molecular weight was 94,088, thenumber-average molecular weight was 81,250, the graft chain molecularweight was 3,250, and the number of graft chains was 21.

As for compound 9, the weight-average molecular weight was 137,695, thenumber-average molecular weight was 109,630, the graft chain molecularweight was 6,442, and the number of graft chains was 15.

Example 3 Preparation Method of Microparticles Encapsulating HumanGrowth Hormone (hGH)

5 mg of dextran-polylactic acid (PLA) of Example 1 (average molecularweight of dextran is 13,000, average molecular weight of PLA is 2,300,number of graft chains of PLA is 10 to 12, compound 3) or dextran-poly(lactic acid-glycolic acid) (PLGA) of Example 2 (average molecularweight of dextran: 13,000, average molecular weight of PLGA is 19,000,number of graft chains of PLGA 7 to 10, compound 7) was dissolved in 100gl of dimethyl carbonate to prepare a polymer solution of 50 mg/ml. Inthis polymer solution, 20 gl of tert-butanol was added, and 20 μl of 2mg/ml hGH aqueous solution was dropped, and stirred by vortex to preparea reversed-phase emulsion. This reversed-phase emulsion was frozenpreliminarily, and was freeze-dried by using a freeze-drying apparatus(EYELA, FREEZE DRYER FD-1000), at trap cooling temperature of −45° C.,and degree of vacuum of 20 Pa, for 24 hours. The obtained solid contentwas dispersed in 200 μl of dimethyl carbonate to prepare an S/Osuspension. This S/O suspension was dropped in 2 ml of aqueous solutioncontaining 10% Pluronic F-68 (a registered trademark of BASF), and wasstirred and emulsified in a vortex mixer to prepare S/O/W type emulsion.From this S/O/W type emulsion, the water-immiscible organic solvent wasremoved by drying in liquid, and a microparticle dispersion liquid wasobtained. The microparticle dispersion liquid was preliminarily frozenby liquid nitrogen, and was freeze-dried by using a freeze-dryingapparatus (EYELA, FREEZE DRYER FD-1000), at trap cooling temperature of−45° C., and degree of vacuum of 20 Pa, for 24 hours, andhGH-encapsulating microparticle powder was obtained. The obtainedmicroparticles were observed by a scanning electron microscope (SEM:HITACHI, S-4800), and the average particle diameter was calculated, andthe average particle diameter of the microparticles was 4.0 μm.

Example 4 Measurement of Drug Encapsulation Efficiency of MicroparticlesEncapsulating Human Growth Hormone (hGH)

20 mg of microparticles encapsulating human growth hormone prepared inthe method of Example 3 by using dextran-PLA (compound 3) ordextran-PLGA (compound 7) polymer was weighed by using a 1.5 mlEppendorf tube, and was dissolved in 1 ml of buffer solution A (PBScontaining 0.1% bovine serum albumin, 0.1% Pluronic F-68 (a registeredtrademark of BASF), and 0.02% sodium azide), and was centrifuged for 10minutes at 18,000×g, and was separated into particles (precipitation)and a supernatant. The supernatant was collected in other tube, and theparticles were suspended again in 1 ml of buffer solution, and thecentrifugal operation and the separation into particles and asupernatant were conducted again in the same conditions. This cleaningoperation was repeated once more (total three times of centrifugaloperation), and the human growth hormone concentration of eachsupernatant collected by the centrifugal operations was measured byusing an ELISA kit (manufactured by R&D Systems). From the chargedamount of hGH at the time of preparation of particles (particle weight20 mg), the hGH total amount of three supernatants by centrifugaloperations was subtracted, and the encapsulation efficiency wascalculated according to the formula below:

$\begin{matrix}{{Encapsulation}\mspace{14mu}} \\{{efficiency}\mspace{14mu} (\%)}\end{matrix} = {\frac{\begin{pmatrix}{{{charged}\mspace{14mu} {hGH}\mspace{14mu} {amount}\mspace{14mu} ({ng})} -} \\{{hGH}\mspace{14mu} {amount}\mspace{14mu} {total}\mspace{14mu} {in}\mspace{14mu} {supernatants}\mspace{14mu} ({ng})}\end{pmatrix}}{{charged}\mspace{14mu} {hGH}\mspace{14mu} {amount}\mspace{14mu} ({ng})} \times 100}$

In dextran-PLA microparticles or dextran-PLGA microparticles, theencapsulation efficiency of hGH was 92.6% in dextran-PLA microparticles,and 85.7% in dextran-PLGA microparticles, and it was proved that theprotein drug can be encapsulated at a high efficiency in both particles.

Example 5 Analysis of In-Vitro Drug Release Speed from MicroparticlesEncapsulating Human Growth Hormone (hGH)

The microparticles centrifuged three times in Example 4 were suspendedand dispersed in 1.2 ml of buffer solution A. From this solution, a part(40 μl) was transferred into other tube, and was centrifuged for 10minutes at 18,000×g to precipitate the particles, and 30 μl ofsupernatant was collected in a different tube (0-hour sample). Theremaining particle suspension was put in a 1.5 ml Eppendorf tube, andwas rolled and mixed slowly in an incubator at 37° C., by using arotator at a speed of 6 rpm. From this solution, a small portion (40 μl)was dispensed at specific time intervals, and the supernatant wasseparated similarly by centrifugal operation. In the supernatant samplecollected at each time, the hGH concentration was measured by using theELISA kit, and the release amount (%) was calculated in the formulabelow:

${{Release}\mspace{14mu} {amount}\mspace{14mu} (\%)} = {\frac{\left( {{hGH}\mspace{14mu} {concentration}\mspace{14mu} {in}\mspace{14mu} {supernatant}\mspace{14mu} \left( {{ng}\text{/}{ml}} \right) \times 1.2\mspace{14mu} ({ml})} \right)}{{encapsulated}\mspace{14mu} {hGH}\mspace{14mu} {amount}\mspace{14mu} ({ng})\mspace{14mu} {in}\mspace{14mu} 20\mspace{14mu} {mg}\mspace{14mu} {of}\mspace{14mu} {particles}} \times 100}$

FIG. 1 shows time-course changes of drug release from microparticlesmanufactured by using dextran-PLA or dextran-PLGA polymer. In bothparticles, initial burst was hardly observed, and the drug was releasedlinearly in proportion to the lapse of time, and a favorable profile wasobserved. The time required for 50% release of the drug was about 1month in the dextran-PLA microparticle, and about 1 week in thedextran-PLGA microparticle, and it was suggested that the release speedcan be controlled by selecting the type of poly (hydroxy acid).

Example 6 Preparation Method of Microparticles Encapsulating HumanInsulin

5 mg of dextran-PLA (average molecular weight of dextran is 13,000,average molecular weight of PLA is 2,300, number of graft chains of PLAis 10 to 12, compound 3) or dextran-PLGA (average molecular weight ofdextran is 13,000, average molecular weight of PLGA is 19,000, number ofgraft chains of PLGA 7 to 10, compound 7) was dissolved in 100 μl ofdimethyl carbonate to prepare a polymer solution of 50 mg/ml. In thispolymer solution, 20 μl of tert-butanol was added, and 20 μl of 2 mg/mlhuman insulin aqueous solution was dropped, and stirred by vortex toprepare a reversed-phase emulsion. This reversed-phase emulsion wasfrozen preliminarily by liquid nitrogen, and was freeze-dried by using afreeze-drying apparatus (EYELA, FREEZE DRYER FD-1000), at trap coolingtemperature of −45° C., and degree of vacuum of 20 Pa, for 24 hours. Theobtained solid content was dispersed in 200 μl of dimethyl carbonate toprepare an S/O suspension. This S/O suspension was dropped in 2 ml ofaqueous solution containing 10% Pluronic F-68 (a registered trademark ofBASF), and was stirred and emulsified in a vortex mixer to prepare anS/O/W type emulsion. From this S/O/W type emulsion, the water-immiscibleorganic solvent was removed by drying in liquid, and a microparticledispersion liquid was obtained. The microparticle dispersion liquid waspreliminarily frozen by liquid nitrogen, and was freeze-dried by using afreeze-drying apparatus (EYELA, FREEZE DRYER FD-1000), at trap coolingtemperature of −45° C., and degree of vacuum of 20 Pa, for 24 hours, andhuman insulin-encapsulating microparticle powder was obtained. Theobtained microparticles were observed by a scanning electron microscope(SEM: HITACHI, S-4800), and the average particle diameter wascalculated, and the average particle diameter was 6.4 μm in themicroparticles obtained from compound 3, and 5.3 μm in themicroparticles obtained from compound 7.

Example 7 Measurement of Drug Encapsulation Efficiency of MicroparticlesEncapsulating Human Insulin

20 mg of microparticles encapsulating human insulin prepared in themethod of Example 6 by using dextran-PLGA (compound 7) polymer wasweighed by using a 1.5 ml Eppendorf tube, and was dissolved in 1 ml ofbuffer solution A (PBS containing 0.1% bovine serum albumin, 0.1%Pluronic F-68 (a registered trademark of BASF), and 0.02% sodium azide),and was centrifuged for 10 minutes at 18,800×g, and was separated intoparticles (precipitation) and a supernatant. The supernatant wascollected in other tube, and the particles were suspended again in 1 mlof buffer solution, and the centrifugal operation and the separationinto particles and a supernatant were conducted again in the sameconditions. This cleaning operation was repeated once more (total threetimes of centrifugal operation), and the human insulin concentration ofeach supernatant collected by the centrifugal operations was measured bysandwich ELISA method. From the charged amount of human insulin at thetime of preparation of particles (per particle weight 20 mg), the humaninsulin total amount of three supernatants by centrifugal operations wassubtracted, and the encapsulation efficiency was calculated according tothe formula below:

$\begin{matrix}{{Encapsulation}\mspace{14mu}} \\{{efficiency}\mspace{14mu} (\%)}\end{matrix} = {\frac{\begin{pmatrix}{{{charged}\mspace{14mu} {insulin}\mspace{14mu} {amount}\mspace{14mu} ({ng})} -} \\{{insulin}\mspace{14mu} {amount}\mspace{14mu} {total}\mspace{14mu} {in}\mspace{14mu} {supernatants}\mspace{14mu} ({ng})}\end{pmatrix}}{{charged}\mspace{14mu} {insulin}\mspace{14mu} {amount}\mspace{14mu} ({ng})} \times 100}$

In dextran-PLA microparticles or dextran-PLGA microparticles, theencapsulation efficiency of human insulin was 75.7%, and it was provedthat the drug can be encapsulated at a high efficiency.

Example 8 Analysis of In-Vitro Drug Release Speed from MicroparticlesEncapsulating Human Insulin

The microparticles centrifuged three times in Example 7 were suspendedand dispersed in 1.2 ml of buffer solution A. From this solution, a part(40 μl) was transferred into other tube, and was centrifuged for 10minutes at 18,800×g to precipitate the particles, and 30 μl ofsupernatant was collected in a different tube (0-hour sample). Theremaining particle suspension was put in a 1.5 ml Eppendorf tube, andwas rolled and mixed slowly in an incubator at 37° C., by using arotator at a speed of 6 rpm. From this solution, a small portion (40 μl)was dispensed at specific time intervals, and the supernatant wasseparated similarly by centrifugal operation. In the supernatant samplecollected at each time, the human insulin concentration was measured bythe sandwich ELISA method, and the release amount (%) was calculated inthe formula below:

${{Release}\mspace{14mu} {amount}\mspace{14mu} (\%)} = {\frac{\left( {{human}\mspace{14mu} {insulin}\mspace{14mu} {concentration}\mspace{14mu} {in}\mspace{14mu} {supernatant}\mspace{14mu} \left( {{ng}\text{/}{ml}} \right) \times 1.2\mspace{14mu} ({ml})} \right)}{{encapsulated}\mspace{14mu} {human}\mspace{14mu} {insulin}\mspace{14mu} {amount}\mspace{14mu} ({ng})\mspace{14mu} {in}\mspace{14mu} 20\mspace{14mu} {mg}\mspace{14mu} {of}\mspace{14mu} {particles}} \times 100}$

FIG. 2 shows time-course changes of human insulin release. Initial burstwas hardly observed, and the drug was released linearly in proportion tothe lapse of time, and a favorable profile was observed. The timerequired for 50% release of the drug was about 6 days.

Example 9 Time-Course Changes of Microparticle Morphology

5 mg of microparticles encapsulating hGH prepared in Example 3 wasweighed in an Eppendorf tube, and dispersed in 1 ml of Milli-Q, and wascentrifugally separated for 30 minutes at 13,000 rpm, and deprived ofthe supernatant, and dispersed again in 1 ml of Milli-Q, andcentrifugally separated, and the microparticles were cleaned. In themicroparticle suspension solution incubated for a specified time, 1 mlof Milli-Q was added, and the solution was centrifugally separated for30 minutes at 13,000 rpm, deprived of the supernatant, and dispersedagain in 1 ml of Milli-Q, and centrifugally separated, and themicroparticles were cleaned. The microparticles obtained after cleaningwere dispersed in 100 μl of Milli-Q, and 3 μl of the microparticledispersion liquid was dropped on a silicon substrate, and let stand atroom temperature for 10 minutes, and dried for 3 hours in a desiccator.Then, using an ion sputtering device (HITACHI, E-1030), platinum wasdeposited on the sample surface (deposition time 15 seconds), and themicroparticle shape and the surface state were observed by a scanningelectron microscope (SEM: HITACHI, S-4800), at an acceleration voltageof 1 kV and a high probe current.

As shown in FIG. 3, right after manufacture, the surface was smooth andspherical, and the particles were obviously deformed after incubationfor 13 days at 37° C., many pores were formed, and it was proved thatthe particles were decomposed gradually along with the progress ofrelease of the drug.

Comparative Example 1

5 mg of polyethylene glycol-poly (epsilon-caprolactone) (averagemolecular weight of polyethylene glycol is 5,000, average molecularweight of poly (epsilon-caprolactone) is 37,000) was dissolved in 100 μlof dimethyl carbonate to prepare a polymer solution of 50 mg/ml. In thispolymer solution, 20 μl of tert-butanol was added, and 20 μl of 2 mg/mlhGH aqueous solution was dropped, and stirred by vortex to prepare areversed-phase emulsion. This reversed-phase emulsion was frozenpreliminarily by liquid nitrogen, and was freeze-dried by using afreeze-drying apparatus (EYELA, FREEZE DRYER FD-1000), at trap coolingtemperature of −45° C., and degree of vacuum of 20 Pa, for 24 hours. Theobtained solid content was dispersed in 200 μl of dimethyl carbonate toprepare an S/O suspension. This S/O suspension was dropped in 2 ml ofaqueous solution containing 10% Pluronic F-68 (a registered trademark ofBASF), and was stirred and emulsified in a vortex mixer to prepare anS/O/W type emulsion. From this S/O/W type emulsion, the water-immiscibleorganic solvent was removed by drying in liquid, and a microparticledispersion liquid was obtained. The microparticle dispersion liquid waspreliminarily frozen by liquid nitrogen, and was freeze-dried by using afreeze-drying apparatus (EYELA, FREEZE DRYER FD-1000), at trap coolingtemperature of −45° C., and degree of vacuum of 20 Pa, for 24 hours, andhGH-encapsulating microparticle powder was obtained. The obtainedmicroparticles were observed by a scanning electron microscope (SEM:HITACHI, S-4800), and the average particle diameter was calculated, andthe average particle diameter of the microparticles was 8.0 μm.

5 mg of the prepared microparticle powder encapsulating hGH was weighedby using an Eppendorf tube, and was dispersed in 1 ml of Milli-Q, andwas centrifuged for 30 minutes at 13,000, deprived of the supernatant,and dispersed again in 1 ml of Milli-Q, and centrifugally separatedsimilarly, and the microparticles were cleaned. The microparticlesobtained after cleaning were dispersed in 100 μl of Milli-Q, and 5 μl ofthe microparticle dispersion liquid was dropped on a silicon substrate,and let stand at room temperature for 10 minutes, and dried for 3 hoursin a desiccator. Then, using an ion sputtering device (HITACHI, E-1030),platinum was deposited on the sample surface (deposition time 15seconds), and the microparticle shape and the surface state wereobserved by a scanning electron microscope (SEM: HITACHI, S-4800), at anacceleration voltage of 1 kV and a high probe current.

As shown in FIG. 4, different from the dextran-PLGA microparticle inExample 9, after incubation for 21 days at 37° C., the particles werehardly changed morphologically, and there was a problem in releasingperformance of hydrophilic active substance.

Example 10 Hypodermic Administration of Microparticles EncapsulatingHuman Growth Hormone (hGH) in Mouse

25 mg of dextran-polylactic acid (PLA) (average molecular weight ofdextran is 13,000, average molecular weight of PLA is 2,300, number ofgraft chains of PLA is 10 to 12, compound 3) or dextran-poly (lacticacid-glycolic acid) (PLGA) (average molecular weight of dextran is13,000, average molecular weight of PLGA 19,000, number of graft chainsof PLGA is 7 to 10, compound 7) was dissolved in 500 μl of dimethylcarbonate to prepare a polymer solution of 50 mg/ml. In this polymersolution, 100 μl of tert-butanol was added, and 250 μl of 10 mg/ml hGHaqueous solution was dropped, and stirred by vortex to prepare areversed-phase emulsion. This reversed-phase emulsion was frozenpreliminarily, and was freeze-dried by using a freeze-drying apparatus(EYELA, FREEZE DRYER FD-1000), at trap cooling temperature of −45° C.,and degree of vacuum of 20 Pa, for 24 hours. The obtained solid contentwas dispersed in 1 ml of dimethyl carbonate to prepare an S/Osuspension. This S/O suspension was dropped in 10 ml of aqueous solutioncontaining 10% Pluronic F-68 (a registered trademark of BASF), and wasstirred and emulsified in a vortex mixer to prepare an S/O/W typeemulsion. From this S/O/W type emulsion, the water-immiscible organicsolvent was removed by drying in liquid, and a microparticle dispersionliquid was obtained. The microparticle dispersion liquid waspreliminarily frozen by liquid nitrogen, and was freeze-dried by using afreeze-drying apparatus (EYELA, FREEZE DRYER FD-1000), at trap coolingtemperature of −45° C., and degree of vacuum of 20 Pa, for 24 hours, andhGH-encapsulating microparticle powder was obtained. The obtainedmicroparticles were observed by a scanning electron microscope (SEM:HITACHI, S-4800), and the average particle diameter was calculated, andthe average particle diameter was 4.9 μm in the microparticles obtainedfrom compound 3, and 4.2 μm in the microparticles obtained from compound7.

300 mg of the prepared microparticles was suspended and dispersed in 3ml of phosphate physiological buffer solution (PBS), and centrifuged for5 minutes at 80×g to precipitate microparticles, and the supernatant wastransferred into other tube. The supernatant was centrifuged again for 5minutes at 80×g to precipitate the remaining particles, and thesupernatant was removed. By re-dispersing in 1 ml of PBS after firsttime of centrifugal precipitation and second time of centrifugalprecipitation, the same centrifugal cleaning operation was repeatedthree times, and the growth hormone not encapsulated in themicroparticles were removed. Finally, the precipitation was dispersedagain in 200 μl of PBS, and an administration solution was obtained. Thegrowth hormone amount encapsulated in dextran-PLA microparticle anddextran-PLGA microcapsule was measured by an ELISA kit and theconcentration in the cleaning solution was determined, and subtractedfrom the charged amount, and the amount encapsulated in 300 mg ofparticles administered per mouse was determined, and the dextran-PLAmicroparticle was 590 μg, and the dextran-PLGA microparticles was 536μg.

This solution was injected hypodermically at two positions in the backof 10-week male Balb/C mouse, and the blood was sampled at specific timeintervals from the caudal vein. In the sampled blood, heparin of finalconcentration of 3.31 U/ml was added, and plasma was collected bycentrifugal separation for 5 minutes at 5,000 rpm, and the concentrationof growth hormone in plasma was measured by using an ELISA kit.

By way of comparison, a non-granulated human growth hormone proteinsolution (700 μg/0.2 ml) was hypodermically administered in mouse, andthe blood was sampled similarly.

To suppress antibody production by administration of human growthhormone, which is a dissimilar protein for mouse, three days beforeadministration of the particle, an immunosuppressant Tacrolimus hydrate(Astellas) was hypodermically administered by 26 μg/mouse, andthereafter 13 μg/mouse was hypodermically administered at the time ofthe drug administration, and 3 days and 7 days later.

FIG. 5 shows time-course changes of concentration of human growthhormone in plasma. In the mouse administered non-granulated drug, theblood level in 1 hour after administration was very high, more than5,000 ng/ml, and then dropped suddenly, to a level before administrationin a day. On the other hand, in the mouse administered the microparticledrug prepared by using dextran-PLA polymer, a transient elevation ofblood level right after administration was suppressed to 200 ng/ml orless, and for seven consecutive days, the blood level was sustained athigh levels. In dextran-PLA microparticles, transient elevation ofconcentration after administration was not observed at all, and a nearlyspecific blood concentration was maintained for seven days, and anexcellent sustained-release performance was observed.

Example 11 Hypodermic Administration of Microparticles EncapsulatingHuman Growth Hormone (hGH) in Mouse (Pharmacological ActivityEvaluation)

2 mg of dextran-poly (lactic acid-glycolic acid) (PLGA) (averagemolecular weight of dextran is 13,000, average molecular weight of PLGAis 1,900, number of graft chains of PLGA is 7 to 10, compound 7) wasdissolved in 500 μl of dimethyl carbonate to prepare a polymer solutionof 50 mg/ml. In this polymer solution, 100 μl of tert-butanol was added,and 250 μl of 10 mg/ml hGH aqueous solution was dropped, and stirred byvortex to prepare a reversed-phase emulsion. This reversed-phaseemulsion was frozen preliminarily by liquid nitrogen, and wasfreeze-dried by using a freeze-drying apparatus (EYELA, FREEZE DRYERFD-1000), at trap cooling temperature of −45° C., and degree of vacuumof 20 Pa, for 24 hours. The obtained solid content was dispersed in 1 mlof dimethyl carbonate to prepare an S/O suspension. This S/O suspensionwas dropped in 10 ml of aqueous solution containing 10% Pluronic F-68 (aregistered trademark of BASF), and was stirred and emulsified in avortex mixer to prepare an S/O/W type emulsion. From this S/O/W typeemulsion, the water-immiscible organic solvent was removed by drying inliquid, and a microparticle dispersion liquid was obtained. Themicroparticle dispersion liquid was preliminarily frozen by liquidnitrogen, and was freeze-dried by using a freeze-drying apparatus(EYELA, FREEZE DRYER FD-1000), at trap cooling temperature of −45° C.,and degree of vacuum of 20 Pa, for 24 hours, and hGH-encapsulatingmicroparticle powder was obtained. The obtained microparticles wereobserved by a scanning electron microscope (SEM: HITACHI, S-4800), andthe average particle diameter was calculated, and the average particlediameter of the obtained microparticles was 4.1 μm.

300 mg of the prepared microparticles was suspended and dispersed in 3ml of phosphate physiological buffer solution (PBS), and particles wereprecipitated by centrifugal separation for 5 minutes at 80×g, and asupernatant was transferred in other tube. The supernatant wascentrifugally separated again for 5 minutes at 80×g, and the remainingparticles were precipitated, and the supernatant was removed. The firstcentrifugal precipitation and the second centrifugal precipitation werecombined, and dispersed again in 1 ml of PBS, and similarly a thirdcentrifugal operation was conducted, and the growth hormone notencapsulated in the particles was removed. Finally, the precipitationwas dispersed again in 200 μl of PBS to prepare an administrationsolution.

This solution was hypodermically injected in the back of 8-week-oldpituitary gland extracted mouse (from Japan SLC), and the blood wassampled at specific intervals from the caudal vein. In the sampledblood, heparin of final concentration of 3.3 IU/ml was added, andcentrifuged for 5 minutes at 5,000 rpm, and the plasma was collected,and the growth hormone concentration in plasma and the mouse IGF-1concentration were measured by ELISA method.

By way of comparison, a non-granulated human growth hormone proteinsolution (700 μg/0.2 ml) was hypodermically administered in mouse, andthe blood was sampled similarly.

To suppress antibody production by administration of human growthhormone, which is a foreign protein for mouse, three days beforeadministration of the particle, an immunosuppressant Tacrolimus hydrate(Astellas) was hypodermically administered by 26 μg/mouse, andthereafter 13 μg/mouse was hypodermically administered at the time ofthe drug administration, and 3 days and 7 days later.

FIG. 6 shows time-course changes of concentration of human growthhormone in plasma. In the mouse administered non-granulated drug, theblood level in 1 hour after administration was very high, and thendropped suddenly, to a level before administration in two days. On theother hand, in the dextran-PLGA microparticle, a transient concentrationelevation right after administration was suppressed low, and for tenconsecutive days after administration, the concentration in plasma wassustained at high levels. At this time, the body weight changes of mouseare shown in FIG. 7. In the mouse administered the growth hormone alone,the body weight increased was suppressed at about 5%, but in the mouseadministered the dextran-PLGA microparticles, the body weight increasedabout 20%.

FIG. 8 shows the IGF-1 concentration in plasma. The IGF-1 concentrationin plasma is correlated with the human growth hormone concentration inblood, and in the mouse administered the dextran-PLGA microparticles,high levels were maintained for ten days after administration.

Example 12 Analysis of Drug Release Speed in Buffer Solution fromMicroparticles Encapsulating Exendin-4 (GLP-1 Receptor Agonist)

25 mg of dextran-poly (lactic acid-glycolic acid) (PLGA) (averagemolecular weight of dextran is 13,000, average molecular weight of PLGAis 3,250 (compound 8) or 6,442 (compound 9), number of graft chains ofPLGA is 21 (compound 8) or 15 (compound 9) was dissolved in 500 μl ofdimethyl carbonate to prepare a polymer solution of 50 mg/ml. In thispolymer solution, 100 μl of tert-butanol was added, and 250 μl of 10mg/ml Exendin-4 (synthesized by commission with Sigma Genosys) wasdropped, and stirred by vortex to prepare a reversed-phase emulsion.This reversed-phase emulsion was frozen preliminarily by liquidnitrogen, and was freeze-dried by using a freeze-drying apparatus(EYELA, FREEZE DRYER FD-1000), at trap cooling temperature of −45° C.,and degree of vacuum of 20 Pa, for 24 hours. The obtained solid contentwas dispersed in 1 ml of dimethyl carbonate to prepare an S/Osuspension. This S/O suspension was dropped in 10 ml of aqueous solutioncontaining 10% Pluronic F-68 (a registered trademark of BASF), and wasstirred and emulsified in a vortex mixer to prepare an S/O/W typeemulsion. From this S/O/W type emulsion, the water-immiscible organicsolvent was removed by drying in liquid, and a microparticle dispersionliquid was obtained. The microparticle dispersion liquid waspreliminarily frozen by liquid nitrogen, and was freeze-dried by using afreeze-drying apparatus (EYELA, FREEZE DRYER FD-1000), at trap coolingtemperature of −45° C., and degree of vacuum of 20 Pa, for 24 hours, andExendin-4-encapsulating microparticle powder was obtained. The obtainedmicroparticles were observed by a scanning electron microscope (SEM:HITACHI, S-4800), and the average particle diameter was calculated, andthe average particle diameter was 4.3 μm in compound 8, and 4.5 μm incompound 9.

These microparticles were cleaned three times according to the method inExample 4, and were suspended and dispersed in 1.2 ml of buffer solutionA. From this solution, a part (40 μl) was transferred into other tube,and was centrifuged for 10 minutes at 18,000×g to precipitate theparticles, and 30 μl of supernatant was collected in a different tube(0-hour sample). The remaining particle suspension was put in a 1.5 mlEppendorf tube, and was rolled and mixed slowly in an incubator at 37°C., by using a rotator at a speed of 6 rpm. From this solution, a smallportion (40 μl) was dispensed at specific time intervals, and thesupernatant was separated similarly by centrifugal operation. In thesupernatant sample collected at each time, the Exendin-4 concentrationwas measured by the ELISA method, and the release amount (%) wascalculated in the formula below:

${{Release}\mspace{14mu} {amount}\mspace{14mu} (\%)} = {\frac{\left( {{Exendin}\text{-}4\mspace{14mu} {concentration}\mspace{14mu} {in}\mspace{14mu} {supernatant}\mspace{14mu} \left( {{ng}\text{/}{ml}} \right) \times 1.2\mspace{14mu} ({ml})} \right)}{{encapsulated}\mspace{14mu} {Exendin}\text{-}4\mspace{14mu} {amount}\mspace{14mu} ({ng})\mspace{14mu} {in}\mspace{14mu} 20\mspace{14mu} {mg}\mspace{14mu} {of}\mspace{14mu} {particles}} \times 100}$

FIG. 9 shows time-course changes of drug release from microparticlesmanufactured by using each dextran-PLGA polymer. In both microparticles,initial burst was hardly observed, and the drug was released linearly inproportion to the lapse of time, and a favorable profile was observed.

Example 13 Hypodermic Administration of Microparticles EncapsulatingExendin-4 (GLP-1 Receptor Agonist) in Mouse

300 mg of microparticles in Example 12 was suspended and dispersed in 3ml of phosphate physiological buffer solution (PBS), and themicroparticles were precipitated by centrifugal operation for 5 minutesat 80×g, and a supernatant was transferred in other tube. Thesupernatant was centrifugally separated again for 5 minutes at 80×g, andthe remaining particles were precipitated, and the supernatant wasremoved. The first centrifugal precipitation and the second centrifugalprecipitation were combined, and dispersed again in 1 ml of PBS, andsimilarly a third centrifugal operation was conducted, and the Exendin-4not encapsulated in the particles was removed. Finally, theprecipitation was dispersed again in 200 μl of PBS to prepare anadministration solution.

This solution was hypodermically injected in the back of 8-week-old SCIDmouse (CB17/lcr-Prkdcscid/CrlCrlk) (from Crea Japan Inc.), and the bloodwas sampled at specific intervals from the caudal vein. In the sampledblood, heparin of final concentration of 3.3 IU/ml was added, andcentrifuged for 5 minutes at 5,000 rpm, and the plasma was collected,and the Exendin-4 concentration in plasma was measured by ELISA method.By way of comparison, a non-granulated Exendin-4 solution (700 μg/0.2ml) was hypodermically administered in mouse, and the blood was sampledsimilarly.

FIG. 10 shows time-course changes of concentration of Exendin-4 inplasma. In the mouse administered non-granulated drug, the blood levelin 1 hour after administration was very high, and then dropped suddenly,to a level before administration. On the other hand, in the dextran-PLGAmicroparticle, a transient concentration elevation after administrationwas suppressed low, and for five consecutive weeks after administration,the concentration in plasma was sustained at high levels.

Example 14 Synthesis of dextran-poly(lactic acid-glycolic acid) (PLGA)14.1 Synthesis of TMS-dextran-PLGA (compound 10, compound 11, compound12, compound 13)

Compound 1 (0.5 g) and tert-butoxy potassium (35 mg) were dried for 1hour at reduced pressure, and tetrahydrofurane (10 ml) was added, andthe mixture was stirred for 1 hour at room temperature. In thissolution, tetrahydrofurane (15 ml) solution of (DL)-lactide (0.558 g)and glycolide (0.45 g) was dropped, and the mixture was stirred for 5minutes. After completion of reaction, the solvent was concentrated atreduced pressure, and purified by reprecipitation by achloroform-methanol system, and TMS-dextran-PLGA (1.96 g) was obtainedas white solid content (compound 10).

In a similar method, by the charging amount of (DL)-lactide (0.67 g) andglycolide (0.54 g), compound 11 was synthesized.

In a similar method, by the charging amount of (DL)-lactide (0.781 g)and glycolide (0.629 g), compound 12 was synthesized.

In a similar method, by the charging amount of (DL)-lactide (1.123 g)and glycolide (0.9 g), compound 13 was synthesized.

14.2 Synthesis of dextran-PLGA (compound 14, compound 15, compound 16,compound 17)

In chloroform solution (10 mL) of compound 10, trifluoroacetic acid (1mL) was added, and stirred for 30 minutes at room temperature. Thesolvent was distilled away at reduced pressure, and the residue wasdissolved in chloroform (10 ml), and dropped into diethyl ether cooledto 0° C., and the product was deposited. The deposition matter wasfiltered away, and concentrated at reduced pressure, and dextran-PLGA(0.44 g) was obtained (compound 14).

From compounds 11, 12, and 13, dextran-PLGA products were obtained by asimilar method (compound 5, compound 16, compound 17). Theweight-average molecular weight and the number-average molecular weightof the polymer of compounds 14 to 17 were determined by GPC measurement(column Toso TSK-gel α-5000×2, DMF system solvent, detector RI, standardproduct, pullulan). The average molecular weight of the graft chain andthe number of graft chains were determined by ¹H-NMR measurement.

As for compound 14, the weight-average molecular weight was 99,462, thenumber-average molecular weight was 85,101, the graft chainnumber-average molecular weight was 2,167, and the number of graftchains was 33.

As for compound 15, the weight-average molecular weight was 107,779, thenumber-average molecular weight was 92,134, the graft chainnumber-average molecular weight was 3,127, and the number of graftchains was 25.

As for compound 16, the weight-average molecular weight was 121,281, thenumber-average molecular weight was 101,873, the graft chainnumber-average molecular weight was 3,000, and the number of graftchains was 30.

As for compound 17, the weight-average molecular weight was 144,838, thenumber-average molecular weight was 122,151, the graft chainnumber-average molecular weight was 4,864, and the number of graftchains was 22.

Example 15 Preparation Method of Microparticles Encapsulating HumanGrowth Hormone (hGH)

5 mg of each dextran-poly (lactic acid-glycolic acid) (dextran-PLGApolymer, compounds 14 to 17) of Example 14 was dissolved in 100 μl ofdimethyl carbonate to prepare a polymer solution of 50 mg/ml. In thispolymer solution, 20 μl of tert-butanol was added, and 50 μl of 1 mg/mlhGH aqueous solution was dropped, and stirred by vortex to prepare areversed-phase emulsion. This reversed-phase emulsion was frozenpreliminarily by liquid nitrogen, and was freeze-dried by using afreeze-drying apparatus (EYELA, FREEZE DRYER FD-1000), at trap coolingtemperature of −45° C., and degree of vacuum of 20 Pa, for 24 hours. Theobtained solid content was dispersed in 200 μl of dimethyl carbonate toprepare an S/O suspension. This S/O suspension was dropped in 2 ml ofaqueous solution containing 10% Pluronic F-68 (a registered trademark ofBASF), and was stirred and emulsified in a vortex mixer to prepare anS/O/W type emulsion. From this S/O/W type emulsion, the water-immiscibleorganic solvent was removed by drying in liquid, and a microparticledispersion liquid was obtained. The microparticle dispersion liquid waspreliminarily frozen by liquid nitrogen, and was freeze-dried by using afreeze-drying apparatus (EYELA, FREEZE DRYER FD-1000), at trap coolingtemperature of −45° C., and degree of vacuum of 20 Pa, for 24 hours, andhGH-encapsulating microparticle powder was obtained. The obtainedmicroparticles were observed by a scanning electron microscope (SEM:HITACHI, S-4800), and the average particle diameter was calculated, andthe average particle diameter of the microparticles was within a rangeof 1.0 to 10 μm.

Example 16 Measurement of Drug Encapsulation Efficiency ofMicroparticles Encapsulating Human Growth Hormone (hGH)

20 mg of microparticles encapsulating human growth hormone prepared inthe method of Example 15 by using each dextran-PLGA polymer (compounds14 to 17) was weighed by using a 1.5 ml Eppendorf tube, and wasdissolved in 1 ml of buffer solution A (PBS containing 0.1% bovine serumalbumin, 0.1% Pluronic F-68 (a registered trademark of BASF), and 0.02%sodium azide), and was centrifuged for 10 minutes at 18,000×g, and wasseparated into particles (precipitation) and a supernatant. Thesupernatant was collected in other tube, and the particles weresuspended again in 1 ml of buffer solution, and the centrifugaloperation and the separation into particles and a supernatant wereconducted again in the same conditions. This cleaning operation wasrepeated once more (total three times of centrifugal operation), and thehuman growth hormone concentration of each supernatant collected by thecentrifugal operations was measured by using an ELISA kit (manufacturedby R&D Systems). From the charged amount of hGH at the time ofpreparation of particles (particle weight 20 mg), the hGH total amountof three supernatants by centrifugal operations was subtracted, and theencapsulation efficiency was calculated according to the formula below:

$\begin{matrix}{{Encapsulation}\mspace{14mu}} \\{{efficiency}\mspace{14mu} (\%)}\end{matrix} = {\frac{\begin{pmatrix}{{{charged}\mspace{14mu} {hGH}\mspace{14mu} {amount}\mspace{14mu} ({ng})} -} \\{{hGH}\mspace{14mu} {amount}\mspace{14mu} {total}\mspace{14mu} {in}\mspace{14mu} {supernatants}\mspace{14mu} ({ng})}\end{pmatrix}}{{charged}\mspace{14mu} {hGH}\mspace{14mu} {amount}\mspace{14mu} ({ng})} \times 100.}$

In dextran-PLGA microparticles, the encapsulation efficiency of hGH was87.5% in microparticles of compound 14, 94.2% in microparticles ofcompound 15, 95.7% in microparticles of compound 16, and 97.5% inmicroparticles of compound 17, and it was proved that the protein drugcan be encapsulated at a high efficiency in all microparticles.

Comparative Example 2 Manufacture of Particles Encapsulating GrowthHormone and Measurement of Drug Encapsulation Efficiency

10 mg dextran-poly (lactic acid-glycolic acid)(PLGA) (compound 14 orcompound 17) was dissolved in 2 mL of ethyl acetate to prepare a polymersolution. In this polymer solution, 100 μL of 0.5 mg/mL hGH aqueoussolution was dropped, and stirred. After stirring operation, thesolution was added to 20 mL of dioxane. The solvent was evaporated, andthe solution was concentrated to about 2 mL, and the particle dispersionliquid was added to water containing 500 mg Pluronic F-68 (a registeredtrademark of BASF). The sample was freeze-dried, and 1 mL of water isadded to 50 mg of the sample, and the particles were dispersed again,and non-associated hydrophilic active substance containing particleswere obtained. The average particle diameter of the particles wasmeasured by a dynamic light scatter method by using an apparatus ELS-Z(manufactured by Otsuka Denshi), and the drug encapsulation efficiencywas determined same as in Example 16.

As a result, in the particles of compound 14, the average particlediameter was 190.5 nm, and the encapsulation efficiency was 73%, and inthe particles of compound 17, the average particle diameter was 197.5nm, and the encapsulation efficiency was 70%, and the encapsulationefficiency was lower than in the microparticles of Example 16.

Example 17 Analysis of In-Vitro Drug Release Speed from MicroparticlesEncapsulating Human Growth Hormone (hGH)

Particles cleaned three times in Example 16 were suspended and dispersedin 1.2 ml of buffer solution A. From this solution, a part (40 μl) wastransferred into other tube, and was centrifuged for 10 minutes at18,000×g to precipitate the particles, and 30 μl of supernatant wascollected in a different tube (0-hour sample). The remaining particlesuspension was put in a 1.5 ml Eppendorf tube, and was rolled and mixedslowly in an incubator at 37° C., by using a rotator at a speed of 6rpm. From this solution, a small portion (40 μl) was dispensed atspecific time intervals, and the supernatant was separated similarly bycentrifugal operation. In the supernatant sample collected at each time,the hGH concentration was measured by the ELISA kit, and the releaseamount (%) was calculated in the formula below:

${{Release}\mspace{14mu} {amount}\mspace{14mu} (\%)} = {\frac{\left( {{hGH}\mspace{14mu} {concentration}\mspace{14mu} {in}\mspace{14mu} {supernatant}\mspace{14mu} \left( {{ng}\text{/}{ml}} \right) \times 1.2\mspace{14mu} ({ml})} \right)}{{encapsulated}\mspace{14mu} {hGH}\mspace{14mu} {amount}\mspace{14mu} ({ng})\mspace{14mu} {in}\mspace{14mu} 20\mspace{14mu} {mg}\mspace{14mu} {of}\mspace{14mu} {particles}} \times 100}$

FIG. 11 shows time-course changes of drug release from microparticlesmanufactured in Example 15. In these microparticles, initial burst washardly observed, and the drug was released linearly in proportion to thelapse of time, and a favorable profile was observed. The time requiredfor 50% release of the drug was about 6 days in microparticles ofcompound 14, about 9 days in microparticles of compound 15, about 16days in microparticles of compound 16, and about 1 month inmicroparticles of compound 17, and it was suggested that the releasespeed could be controlled by changing the charged amount of lactide andglycolide at the time of synthesis of TMS-dextran-PLGA.

Example 18 Preparation Method of Microparticles EncapsulatingFluoresceine Labeled Dextran (FD40) Different in Particle Diameter

5 mg of dextran-poly (lactic acid-glycolic acid) (PLGA) (compound 7) ofExample 2 was dissolved in 100 μl of dimethyl carbonate to prepare apolymer solution of 50 mg/ml. In this polymer solution, 20 μl oftert-butanol was added, and 20 μl of 1 mg/ml FD40 aqueous solution wasdropped, and stirred by vortex to prepare a reversed-phase emulsion.This reversed-phase emulsion was frozen preliminarily by liquidnitrogen, and was freeze-dried by using a freeze-drying apparatus(EYELA, FREEZE DRYER FD-1000), at trap cooling temperature of −45° C.,and degree of vacuum of 20 Pa, for 24 hours. The obtained solid contentwas dispersed in 50 μl, 100 μl, 200 μl, 350 μl, 500 μl, 1 ml, 2 ml, and6 ml of dimethyl carbonate to prepare an S/O suspension. This S/Osuspension was dropped in 2 ml of aqueous solution containing 10%Pluronic F-68 (a registered trademark of BASF), and was stirred andemulsified in a vortex mixer to prepare an S/O/W type emulsion. Fromthis S/O/W type emulsion, the water immiscible organic solvent wasremoved by drying in liquid, and a microparticle dispersion liquid wasobtained. The microparticle dispersion liquid was preliminarily frozenby liquid nitrogen, and was freeze-dried by using a freeze-dryingapparatus (EYELA, FREEZE DRYER FD-1000), at trap cooling temperature of−45° C., and degree of vacuum of 20 Pa, for 24 hours, andFD40-encapsulating microparticle powder was obtained. The obtainedmicroparticles were observed by a scanning electron microscope (SEM:HITACHI, S-4800), and the average particle diameter was calculated.

FIG. 12 shows the correlation between the average particle diameter andthe amount of dimethyl carbonate added at the time of preparation ofS/O/W type emulsion. In a range from 50 μl to 500 μl, along withincrease of dimethyl carbonate amount, decline of the average particlediameter was observed. From 500 μl to 6 ml, almost no difference wasobserved in the average particle diameter.

Example 19 Synthesis of PEG-PLGA Polymer (PEG2k Series)

Polyethylene glycol monomethyl ether (manufactured by NOF Corp.,SUNBRIGHT MEH-20H, number-average molecular weight: 1,862, Mw/Mn=1.03),(DL)-lactide, and glycolide were mixed in the specified compositionshown in Table 1, and heated at 140° C. After stirring for 20 minutes,tin octylate(II) was added (by 0.05 wt. % to polyethylene glycolmonomethyl ether), and stirred for 3 hours at 180° C. The reactionsolution was returned to room temperature, and was dissolved inchloroform (to a concentration of about 100 mg/ml), and precipitatedagain and refined in diethyl ether cooled at 0° C., and the obtainedsolid content was filtered, decompressed, and dried, and PEG-PLGApolymer was obtained as white or pale brown solid content. Thenumber-average molecular weight of this polymer was determined by ¹H-NMR(Table 1).

TABLE 1 Raw material charged amount and reaction results of synthesis ofPEG-PLGA polymer (PEG2k series) Polymer composition (molecular weight ofCharged amount (g) Molecular PEG)-(molecular PEG (DL)-lactide GlycolideYield (g) weight (¹H-NMR) weight of PLGA) 0.8 1.44 1.16 4.74 135002k-11.5k 0.4 1.44 1.16 2.65 23560 2k-21.5k 0.2 1.44 1.16 2.52 527002k-50.7k

Example 20 Synthesis of PEG-PLGA Polymer (PEG5k Series)

Polyethylene glycol monomethyl ether (manufactured by NOF Corp.,SUNBRIGHT MEH-20H, number-average molecular weight: 5,128, Mw/Mn=1.02),(DL)-lactide, and glycolide were mixed in the specified compositionshown in Table 2, and heated at 140° C. After stirring for 20 minutes,tin octylate(II) was added (by 0.05 wt. % to polyethylene glycolmono-methyl ether), and stirred for 3 hours at 180° C. The reactionsolution was returned to room temperature, and was dissolved inchloroform (to a concentration of about 100 mg/ml), and precipitatedagain and refined in diethyl ether cooled at 0° C., and the obtainedsolid content was filtered, decompressed, and dried, and PEG-PLGApolymer was obtained as white or pale brown solid content. Thenumber-average molecular weight of this polymer was determined by ¹H-NMR(Table 2).

TABLE 2 Raw material charged amount and reaction results of synthesis ofPEG-PLGA polymer (PEG5k series) Polymer composition (molecular weight ofCharged amount (g) Molecular PEG)-(molecular PEG (DL)-lactide GlycolideYield (g) weight (¹H-NMR) weight of PLGA) 0.5 0.72 0.58 1.31 156005k-10k 0.5 1.44 1.16 2.51 28400 5k-23k 0.33 1.44 1.16 2.1 37500 5k-32.5k0.6 2.88 2.32 5.5 44400 5k-39.4k 0.27 1.44 1.16 2.62 52000 5k-47k 0.21.44 1.16 2.52 66000 5k-61k 0.3 2.16 1.74 4.07 69935 5k-65k 0.8 2.161.74 3.79 59555 5k-55k 0.1 1.15 0.93 — 109381 5k-105k

Example 21 Synthesis of PEG-PLGA Polymer (PEG10k Series)

Polyethylene glycol monomethyl ether (manufactured by NOF Corp.,SUNBRIGHT MEH-10H, number-average molecular weight: 9,975, Mw/Mn=1.02),(DL)-lactide, and glycolide were mixed in the specified compositionshown in Table 3, and heated at 140° C. After stirring for 20 minutes,tin octylate(II) was added (by 0.05 wt. % to polyethylene glycolmonomethyl ether), and stirred for 3 hours at 180° C. The reactionsolution was returned to room temperature, and was dissolved inchloroform (to a concentration of about 100 mg/ml), and precipitatedagain and refined in diethyl ether cooled at 0° C., and the obtainedsolid content was filtered, decompressed, and dried, and PEG-PLGApolymer was obtained as white or pale brown solid content. Thenumber-average molecular weight of this polymer was determined by ¹H-NMR(Table 3).

TABLE 3 Raw material charged amount and reaction results of synthesis ofPEG-PLGA polymer (PEG10k series) Polymer composition (molecular weightof Charged amount (g) Molecular PEG)-(molecular PEG (DL)-lactideGlycolide Yield (g) weight (¹H-NMR) weight of PLGA) 0.5 1.44 1.16 2.349000 10k-39k 0.25 1.44 1.16 2.48 105000 10k-95k

Example 22 Preparation Method of FD40-Encapsulating Microparticles

5 mg of PEG-PLGA polymer prepared in Examples 19 to 21 was dissolved in100 μl of dimethyl carbonate to prepare a polymer solution of 50 mg/ml.In this polymer solution, 20 μl of tert-butanol was added, a specifiedamount of 10 mg/ml FD40 aqueous solution as shown in Table 4 was added,and stirred to prepare a reversed-phase emulsion. This reversed-phaseemulsion was frozen preliminarily by liquid nitrogen, and wasfreeze-dried by using a freeze-drying apparatus (EYELA, FREEZE DRYERFD-1000), at trap cooling temperature of −45° C., and degree of vacuumof 20 Pa, for 24 hours. The obtained solid content was dispersed in 200μl of dimethyl carbonate to prepare an S/O suspension. This S/Osuspension was dropped in 2 ml of aqueous solution containing 10%Pluronic F-68 (a registered trademark of BASF), and was stirred andemulsified in a vortex mixer to prepare an S/O/W type emulsion. Fromthis S/O/W type emulsion, the water-immiscible organic solvent wasremoved by drying in liquid, and a microparticle dispersion liquid wasobtained. The microparticle dispersion liquid was preliminarily frozenby liquid nitrogen, and was freeze-dried by using a freeze-dryingapparatus (EYELA, FREEZE DRYER FD-1000), at trap cooling temperature of−45° C., and degree of vacuum of 20 Pa, for 24 hours, andFD40-encapsulating microparticle powder was obtained, and a part thereofwas observed by a scanning electron microscope (SEM: HITACHI, S-4800),and the average particle diameter was calculated (Table 4). SEM imagesof the powder prepared from the PEG-PLGA polymer of 5k to 10k are shownin FIG. 14, and SEM images of the powder prepared from the PEG-PLGApolymer of 5k to 61k are shown in FIG. 15.

TABLE 4 FD40 aqueous solution amount to be added to each polymer, andaverage particle diameter of obtained microparticles. PEG-PLGA FD40aqueous Average particle composition solution amount (μL) diameter (μm)2k-11.5k 13 — 2k-21.5k 12 — 2k-50.7k 12 — 5k-10k   20 — 5k-23k   20 4.65k-32.5k 20 4.3 5k-39.4k 20 — 5k-47k   20 4.2 5k-61k   20 3.9 5k-65k  20 3.2 10k-39k   18 4.8 10k-95k   15 4.5

Example 23 Measurement of Encapsulation Efficiency of FD40-EncapsulatingMicroparticles

Microparticle (5 mg) encapsulating FD40 prepared in the method ofExample 22 by using the PEG-PLGA polymer was weighed by using a 1.5 mlEppendorf tube, and was dispersed in Milli-Q (1 ml), and centrifuged for30 minutes, and separated into a supernatant containing non-encapsulatedFD40 and FD40-encapsulating particles, and collected. The collectedFD40-encapsulating particles were dissolved in N,N-dimethyl formamide(250 μl), and the particles were disintegrated. The supernatantcontaining non-encapsulated FD40 and N,N-dimethyl formamide solution (50μl) containing encapsulated FD40 were added to Milli-Q (3 ml)individually, and stirred well, and FD40 was quantitatively determinedby using a fluorescent spectrophotometer (HORIBA, Fluoro MAX-3,excitation wavelength 495 nm, fluorescent wavelength 520 nm), and theencapsulation efficiency in the whole collection volume was calculated.

FIG. 13 shows the encapsulation efficiency of FD40 in microparticlesprepared from PEG-PLGA polymer. In all series of 2k, 5k, 10k ofmolecular weight of PEG, when the molecular weight of PLG was high, theencapsulation efficiency tended to be high. In particular, in the PEG5kseries, at 5k-65k, the encapsulation efficiency was very high, beingabout 90%. The encapsulation efficiency was about 55% in 10k-95k(PLGA/PEG=9.5) nearly at a same molecular weight ratio as 5k-47k*OKGA/PEG=9.4) of high encapsulation efficiency (about 80%), theencapsulation efficiency was about 55%.

Comparative Example 3 Manufacture of Particles Encapsulating FD40

10 mg of PEG-PLGA polymer (5k-61k) was dissolved in 2 mL of ethylacetate to prepare a polymer solution. In this polymer solution, 100 μLof 2 mg/mL growth hormone solution was dropped, and stirred. Afterstirring operation, the solution was added to 20 mL of dioxane. Thesolvent was evaporated, and concentrated to about 2 mL, and the particledispersion liquid was added to water containing 500 mg Pluronic F-68 (aregistered trademark of BASF). The sample was freeze-dried, and 1 mL ofwater is added to 50 mg of the sample, and the particles were dispersedagain, and non-associated hydrophilic active substance containingparticles were obtained. The average particle diameter of the particleswas measured by a dynamic light scatter method by using an apparatusELS-Z (manufactured by Otsuka Denshi), and the drug encapsulationefficiency was determined same as in Example 23.

As a result, the encapsulation efficiency of FD40 was 48%, the averageparticle diameter was 203.8 nm, and the encapsulation efficiency waslower than in the microparticles of Example 23.

Example 24 Analysis of In-Vitro FD40 Release Speed from MicroparticlesEncapsulating FD40

To evaluate the relation between the sustained-release behavior and thelength of PLGA chain for composing the PEG-PLGA polymer particle,release behavior was evaluated in particles of 5k-23k, 5k-32.5k, 5k-47k,and 5k-61k, out of the microparticles encapsulating the FD40 prepared inExample 22.

Microparticles were, right after preparation, stored in freeze-driedstate at −30° C., and returned to normal temperature before use. Exactly20 mg of particle powder was weighed, and put in a 1.5 ml tube(Eppendorf tube), and 1 ml of assay buffer was added (0.02% sodiumazide, 0.1% Pluronic F-68 (a registered trademark of BASF), and 0.1%bovine serum albumin added PBS solution), and stirred firmly by a touchmixer and suspended. Then, using Hitachi high-speed centrifugal machine(CF16RX), the solution was centrifuged for 10 minutes at 18,900×g, and950 μl of supernatant fraction containing non-encapsulated FD40 wasremoved, and 950 μl of assay buffer was added again, and the particleswere suspended and centrifuged, and the particle cleaning operation wasrepeated in a total of three times.

In the particles cleaned three times, 950 μl of assay buffer was addedonce more, and the particles were suspended, and 100 μl each wasdispensed in a 1.5 ml tube. In each tube, 900 μl of assay buffer wasadded to obtain a total solution of 1 ml, which was incubated in anincubator at 37° C. while being rotated at 10 rpm by means of a rotator.Each incubated tube was centrifuged sequentially for 10 minutes at18900×g, and 950 μl of supernatant was dispended, and stored at 4° C.until the time of measurement of fluorescent intensity.

The fluorescent intensity of the sampled solution was measured by using3 ml disposal cuvette (KARTELL) and HORIBA Fluoro MAX-3, at excitationwavelength of 494 nm and fluorescent wavelength of 512 nm, and thesustained-release ratio was determined from the ratio of the amount ofFD40 used in preparation of particles.

FIG. 16 shows the release amount of FD40 from the various microparticlesdetermined by the release evaluation. The axis of abscissas denotes theincubation time, and the axis of ordinates represents the release ratioto the charged amount. In 5k-23k particles short in the PLGA chain,about 40% of the charged amount was released within 1 day in the initialperiod of incubation, and in one month, almost all amount was releasedexcept of the portion of initial burst. By contrast, as the length ofthe PLGA chain becomes longer, the initial release amount decreased, andin microparticles of 5k-61k, the release amount in a first day ofinitial period of 10% or less.

Example 25 Measurement of Drug Encapsulation Efficiency ofMicroparticles Encapsulating Human Insulin

Using the PEG-PLGA polymer (5k-61k) prepared in Example 20,microparticles encapsulating human insulin were prepared in the samemethod as in Example 22. Obtained microparticles (20 mg) were weighed byusing a 1.5 ml Eppendorf tube, and dissolved in 1 ml of buffer solutionA (PBS containing 0.1% bovine serum albumin, 0.1% Pluronic F-68 (aregistered trademark of BASF), and 0.02% sodium azide), and werecentrifuged for 10 minutes at 18,800×g, and separated into particles(precipitation) and a supernatant. The supernatant was collected inother tube, and the particles were suspended again in 1 ml of buffersolution A, and the centrifugal operation and the separation intoparticles and a supernatant were conducted again in the same conditions.This cleaning operation was repeated once more (total three times ofcentrifugal operation), and the human insulin concentration of eachsupernatant collected by the centrifugal operations was measured bysandwich ELISA method.

The sandwich ELISA method was conducted in the following procedure.Anti-human insulin monoclonal antibody (manufactured by Fitzgerald,clone No. E6E5) was immobilized on an ELISA plate (Maxisorp of NuncCorp.) at concentration of 5 μg/ml, and 50 μL of ELISA buffer solution(0.1 M Tris chlorate buffer solution containing 0.25% BSA and 0.05%Tween 20, pH 8.0) and 50 μL of measurement sample or standard samplediluted in ELISA diluting solution (PBS containing 0.25% BSA and 0.05%Tween 20) were added, and the solution was reacted at room temperatureby shaking for 1 hour. The plate was cleaned three times in a cleaningsolution (PBS containing 0.05% Tween 20), and the unreacted reagent wasremoved, and 0.5 μg/ml of biotin-labeled antihuman monoclonal antibody(manufactured by Fitzgerald, clone No. D4B8), and strepto-avidin-HRPconjugate (manufactured by Zymed) were added, and the solutions werereacted at room temperature by shaking for 1 hour and 15 minutes. Aftereach reaction, the plate was cleaned three times in a cleaning solution(PBS containing 0.05% Tween 20), and the unreacted reagent was removed.Finally, the substrate of HRP was added, and the HRP enzyme activity ofthe combined conjugate was determined by colorimetry, and on the basisof the working curve prepared from color development of standardinsulin, the insulin concentration in the sample was determined.

From the charged amount of human insulin at the time of preparation ofparticles (per particle weight 20 mg), the human insulin total amount ofthree supernatants by centrifugal operations was subtracted, and theencapsulation efficiency was calculated according to the formula below:

$\begin{matrix}{{Encapsulation}\mspace{14mu}} \\{{efficiency}\mspace{14mu} (\%)}\end{matrix} = {\frac{\begin{pmatrix}{{{charged}\mspace{14mu} {insulin}\mspace{14mu} {amount}\mspace{14mu} ({ng})} -} \\{{insulin}\mspace{14mu} {amount}\mspace{14mu} {total}\mspace{14mu} {in}\mspace{14mu} {supernatants}\mspace{14mu} ({ng})}\end{pmatrix}}{{charged}\mspace{14mu} {insulin}\mspace{14mu} {amount}\mspace{14mu} ({ng})} \times 100}$

The average particle diameter of the obtained microparticles was 4.7 μm.The encapsulation efficiency of human insulin in microparticles was86.75, and it was proved that the protein drug could be contained at ahigh efficiency.

Example 26 Analysis of In-Vitro Drug Release Speed from MicroparticlesEncapsulating Human Insulin

The microparticles centrifuged three times in Example 25 were suspendedand dispersed in 1.0 ml of buffer solution A. From this solution, 0.1 mleach was dispensed in ten Eppendorf tubes (1.5 ml capacity), and 0.9 mlof buffer solution A was added in each tube, and diluted 10 times. Rightafter dilution, one tube was centrifuged for 10 minutes at 18,800×g toprecipitate the particles, and a supernatant was collected in adifferent tube (0-hour sample). The remaining nine tubes were rolled andmixed slowly in an incubator at 37° C., by using a rotator at a speed of6 rpm. At specific time intervals, each tube was similarly centrifuged,and the supernatant was separated. In the supernatant sample collectedat each time, the insulin concentration was measured by the sandwichELISA method, and the insulin release amount (%) was calculated in theformula below:

${{Release}\mspace{14mu} {amount}\mspace{14mu} (\%)} = {\frac{\left( {{insulin}\mspace{14mu} {concentration}\mspace{14mu} {in}\mspace{14mu} {supernatant}\mspace{14mu} \left( {{ng}\text{/}{ml}} \right) \times 1\mspace{14mu} ({ml})} \right)}{{encapsulated}\mspace{14mu} {insulin}\mspace{14mu} {amount}\mspace{14mu} ({ng})\mspace{14mu} {in}\mspace{14mu} 20\mspace{14mu} {mg}\mspace{14mu} {of}\mspace{14mu} {particles}} \times 100.}$

FIG. 17 shows time-course changes of insulin release. Along with thelapse of time, the drug was released gradually, and the release speedincreased after 30 days, and the majority of the drug was released inabout 60 days.

Example 27 Hypodermic Administration of Microparticles EncapsulatingHuman Growth Hormone (hGH) in Mouse

25 mg of PEG-PLGA polymer was dissolved in 500 μl of dimethyl carbonateto prepare a polymer solution of 50 mg/ml. In this polymer solution, 100μl of tert-butanol was added, and 250 μl of 10 mg/ml hGH aqueoussolution was dropped, and stirred by vortex to prepare a reversed-phaseemulsion. This reversed-phase emulsion was frozen preliminarily byliquid nitrogen, and was freeze-dried by using a freeze-drying apparatus(EYELA, FREEZE DRYER FD-1000), at trap cooling temperature of −45° C.,and degree of vacuum of 20 Pa, for 24 hours. The obtained solid contentwas dispersed in 1 ml of dimethyl carbonate to prepare an S/Osuspension. This S/O suspension was dropped in 10 ml of aqueous solutioncontaining 10% Pluronic F-68 (a registered trademark of BASF), and wasstirred and emulsified in a vortex mixer to prepare an S/O/W typeemulsion. From this S/O/W type emulsion, the water-immiscible organicsolvent was removed by drying in liquid, and a microparticle dispersionliquid was obtained. The microparticle dispersion liquid waspreliminarily frozen by liquid nitrogen, and was freeze-dried by using afreeze-drying apparatus (EYELA, FREEZE DRYER FD-1000), at trap coolingtemperature of −45° C., and degree of vacuum of 20 Pa, for 24 hours, andhGH-encapsulating microparticle powder was obtained. The averageparticle diameter of the obtained microparticles was 6.0 μm.

300 mg of the prepared microparticles was suspended and dispersed in 3ml of phosphate physiological buffer solution (PBS), and centrifuged for5 minutes at 80×g to precipitate microparticles, and a supernatant wastransferred into other tube. The supernatant was centrifuged again for 5minutes at 80×g to precipitate the remaining particles, and thesupernatant was removed. The first centrifugal precipitation and thesecond centrifugal precipitation were combined, and dispersed again in 1ml of PBS, and the same centrifugal cleaning operation was repeatedthree times in total, and the growth hormone not encapsulated in themicroparticles were removed. Finally, the precipitation was dispersedagain in 200 μl of PBS, and an administration solution was obtained. Thegrowth hormone amount encapsulated in PEG-PLGA particles was measured byan ELISA kit, and subtracted from the charged amount, and the amountencapsulated in 300 mg of particles administered per mouse wasdetermined, and 700 μg of PEG-PLGA microparticles was obtained.

This solution was injected hypodermically at two positions in the backof 10-week male Balb/C mouse, and the blood was sampled at specific timeintervals from the caudal vein. In the sampled blood, heparin of finalconcentration of 3.3 IU/ml was added, and plasma was collected bycentrifugal separation for 5 minutes at 5,000 rpm, and the concentrationof growth hormone in plasma was measured by using an ELISA kit.

By way of comparison, a non-granulated human growth hormone proteinsolution (700 μg/0.2 ml) was hypodermically administered in mouse, andthe blood was sampled similarly.

To suppress antibody production by administration of human growthhormone, which is a foreign protein for mouse, three days beforeadministration of the particle, an immunosuppressant Tacrolimus hydrate(Astellas) was hypodermically administered by 26 μg/mouse, andthereafter 13 μg/mouse was hypodermically administered at the time ofthe drug administration, and 3 days and 7days later.

FIG. 18 shows time-course changes of concentration of human growthhormone in plasma. In the mouse administered non-granulated drug, theblood level in 1 hour after administration was very high, more than5,000 ng/ml, and then dropped suddenly, to a level before administrationin a day. On the other hand, in the mouse administered the microparticledrug prepared by using PEG-PLGA polymer, a transient elevation of bloodlevel right after administration was suppressed to 100 ng/ml or less,and for seven consecutive days, the blood level was sustained at highlevels.

Example 28 Manufacture of Microparticles Adding Salt to Liquid Phase inStep (c)

In 100 μl of 50 mg/ml PEG-PLGA polymer (5k-61k)/dimethyl carbonatesolution, 20 μl of tert-butanol was added, and 20 μl of 10 mg/ml FD40aqueous solution was added, and the mixture was stirred to prepare areversed micelle (W/O emulsion) solution. The obtained solution wasfrozen preliminarily by liquid nitrogen, and was freeze-dried overnightby using a freeze-drying apparatus, and a solid content containing FD40was obtained. In the obtained solid content containing FD40, 200 μl ofdimethyl carbonate was added, and stirred for 10 second by vortex toprepare an S/O suspension, and it was dropped in 2 ml of aqueoussolution containing 10% Pluronic F-68 (a registered trademark of BASF)together with sodium chloride at specified concentration (0 M, 10 mM, 50mM, 1 M), and was stirred and emulsified by vortex for 30 seconds toprepare an S/O/W type emulsion solution. From the obtained S/O/W typeemulsion solution, the water-immiscible organic solvent was removed byusing an evaporator (evacuated to 30 hPa, and evacuated and distilledaway for 5 minutes) to prepare a water disperse matter of microparticlescontaining FD40. The disperse aqueous solution of microparticlescontaining FD40 was frozen preliminarily by liquid nitrogen, and wasfreeze-dried overnight by using a freeze-drying apparatus, and FD40containing microparticle powder was obtained. The obtainedmicroparticles were observed by a scanning electron microscope (SEM:HITACHI, S-4800), and the average particle diameter was calculated, andin all sodium chloride concentration conditions, the average particlediameter of microparticles was 6.5 μm.

20 mg of the obtained FD40 containing microparticle powder was weighed,and dispersed in 1 ml of PBS buffer solution (containing 0.1% PluronicF-68 (a registered trademark of BASF), 0.1% BSA, and 0.01% sodiumazide), and centrifuged (14,000 rpm, 10 minutes). After collection ofthe supernatant, the microparticles were suspended again in 1 ml of PBSbuffer solution, and centrifuged, and the microparticles were cleanedfurther two more times. The cleaned microparticles were suspended againin 1 ml of PBS buffer solution, dispended by 900 μl each in 1.5 mlEppendorf tubes, and 900 μl of PBS buffer solution was added, and thesolution was incubated at 37° C., and samples were collected after 24hours. The collected samples were centrifuged for 10 minutes at 14,000rpm, and FD40 contained in the supernatant was measured by using afluorescent spectrophotometer (HORIBA, Fluoro MAX-3, excitationwavelength 495 nm, fluorescent wavelength 520 nm), and the releaseamount was calculated. The amount of FD40 in the supernatant collectedat the time of cleaning was measured similarly, and the encapsulationefficiency was calculated from the charged amount.

The encapsulation efficiency was 73%, 97%, 84%, and 82% at sodiumchloride concentrations of 0 M, 10 mM, 50 mM, and 1 M. The releaseamount in 1 day was 14%, 7%, 15%, and 11% at sodium chlorideconcentrations of 0 M, 10 mM, 50 mM, and 1 M, and at the sodium chlorideconcentration of 10 mM, the encapsulation efficiency was highest, andthe release amount in 1 day (initial burst) was least.

Example 29 Hypodermic Administration of Microparticles EncapsulatingHuman Growth Hormone (hGH) in Mouse (Pharmacological ActivityEvaluation)

25 mg each of PEG-PLGA polymer (5k-55k) and PEG-PLGA polymer (5k-105k)of Example 20 was dissolved in 500 μl of dimethyl carbonate to prepare apolymer solution of 50 mg/ml. In this polymer solution, 100 μl oftert-butanol was added, and 250 μl of 10 mg/ml hGH aqueous solution wasdropped, and stirred by vortex to prepare a reversed-phase emulsion.This reversed-phase emulsion was frozen preliminarily by liquidnitrogen, and was freeze-dried by using a freeze-drying apparatus(EYELA, FREEZE DRYER FD-1000), at trap cooling temperature of −45° C.,and degree of vacuum of 20 Pa, for 24 hours. The obtained solid contentwas dispersed in 1 ml of dimethyl carbonate to prepare an S/Osuspension. This S/O suspension was dropped in 10 ml of aqueous solutioncontaining 10% Pluronic F-68 (a registered trademark of BASF), and wasstirred and emulsified in a vortex mixer to prepare an S/O/W typeemulsion. From this S/O/W type emulsion, the water-immiscible organicsolvent was removed by drying in liquid, and a microparticle dispersionliquid was obtained. The microparticle dispersion liquid waspreliminarily frozen by liquid nitrogen, and was freeze-dried by using afreeze-drying apparatus (EYELA, FREEZE DRYER FD-1000), at trap coolingtemperature of −45° C., and degree of vacuum of 20 Pa, for 24 hours, andhGH-encapsulating microparticle powder was obtained. The obtainedmicroparticles were observed by a scanning electron microscope (SEM:HITACHI, S-4800), and the average particle diameter was calculated, andthe average particle diameter of the obtained microparticles was 4.2 μmin the microparticles from PEG-PLGA polymer (5k-55k) (5k-55kmicroparticles), and 7.5 μm in the microparticles from PEG-PLGA polymer(5k-105k) (5k-105k microparticles).

300 mg each of the microparticles prepared above was suspended anddispersed in 3 ml of phosphate physiological buffer solution (PBS), andparticles were precipitated by centrifugal separation for 5 minutes at80×g, and a supernatant was transferred in other tube. The supernatantwas centrifugally separated again for 5 minutes at 80×g, and theremaining particles were precipitated, and the supernatant was removed.The first centrifugal precipitation and the second centrifugalprecipitation were combined, and dispersed again in 1 ml of PBS, andsimilarly a third centrifugal operation was conducted, and the growthhormone not encapsulated in the particles was removed. Finally, theprecipitation was dispersed again in 200 μl of PBS to prepare anadministration solution.

This solution was hypodermically injected in the back of 8-week-oldpituitary gland extracted ICR mouse (from Japan SLC), and the blood wassampled at specific intervals from the caudal vein. In the sampledblood, heparin of final concentration of 3.3 IU/ml was added, andcentrifuged for 5 minutes at 5,000 rpm, and the plasma was collected,and the growth hormone concentration in plasma and the mouse IGF-1concentration were measured by ELISA method.

By way of comparison, a non-granulated human growth hormone proteinsolution (700 μg/0.2 ml) was hypodermically administered in mouse, andthe blood was sampled similarly.

To suppress antibody production by administration of human growthhormone, which is a foreign protein for mouse, three days beforeadministration of the particle, an immunosuppressant Tacrolimus hydrate(Astellas) was hypodermically administered by 26 μg/mouse, andthereafter 13 μg/mouse was hypodermically administered at the time ofthe drug administration, and twice a week thereafter.

FIG. 19 shows time-course changes of concentration of human growthhormone in plasma. In the mouse administered non-granulated drug, theblood level in 1 hour after administration was very high, and thendropped suddenly, to a level before administration in one day. On theother hand, in the mouse administered the microparticle drugmanufactured by using PEG-PLGA polymer, a transient concentrationelevation right after administration was suppressed low, about 1/100 ofthe level in the mouse administered non-granulated drug, and for morethan nine consecutive days after administration, the blood level wassustained at high levels.

FIG. 20 shows the IGF-1 concentration in plasma during this time period.The IGF-1 concentration in plasma was elevated after administration inboth 5k-55k microparticles and 5k-105k microparticles, and high levelswere maintained for 7 days in 5k-55k microparticles, and more than 14days in 5k-105k microparticles.

Example 30 Hypodermic Administration of Microparticles EncapsulatingExendin-4 (GLP-1 Receptor Agonist) in Mouse

25 mg of PEG-PLGA polymer (5k-61k) in Example 20 was dissolved in 500 μlof dimethyl carbonate to prepare a polymer solution of 50 mg/ml. In thispolymer solution, 100 μl of tert-butanol was added, and 250 μl of 10mg/ml Exendin-4 (synthesized by commission with Sigma Genosys) wasdropped, and stirred by vortex to prepare a reversed-phase emulsion.This reversed-phase emulsion was frozen preliminarily by liquidnitrogen, and was freeze-dried by using a freeze-drying apparatus(EYELA, FREEZE DRYER FD-1000), at trap cooling temperature of −45° C.,and degree of vacuum of 20 Pa, for 24 hours. The obtained solid contentwas dispersed in 1 ml of dimethyl carbonate to prepare an S/Osuspension. This S/O suspension was dropped in 10 ml of aqueous solutioncontaining 10% Pluronic F-68 (a registered trademark of BASF), and wasstirred and emulsified in a vortex mixer to prepare an S/O/W typeemulsion. From this S/O/W type emulsion, the water-immiscible organicsolvent was removed by drying in liquid, and a microparticle dispersionliquid was obtained. The microparticle dispersion liquid waspreliminarily frozen by liquid nitrogen, and was freeze-dried by using afreeze-drying apparatus (EYELA, FREEZE DRYER FD-1000), at trap coolingtemperature of −45° C., and degree of vacuum of 20 Pa, for 24 hours, andExendin-4-encapsulating microparticle powder was obtained. The obtainedmicroparticles were observed by a scanning electron microscope (SEM:HITACHI, S-4800), and the average particle diameter was calculated, andthe average particle diameter of microparticles was 6.0 μm.

300 mg of the prepared microparticles was suspended and dispersed in 3ml of phosphate physiological buffer solution (PBS), and particles wereprecipitated by centrifugal separation for 5 minutes at 80×g, and asupernatant was transferred in other tube. The supernatant wascentrifugally separated again for 5 minutes at 80×g, and the remainingparticles were precipitated, and the supernatant was removed. The firstcentrifugal precipitation and the second centrifugal precipitation werecombined, and dispersed again in 1 ml of PBS, and similarly a thirdcentrifugal operation was conducted, and the Exendin-4 not encapsulatedin the particles was removed. Finally, the precipitation was dispersedagain in 200 μl of PBS to prepare an administration solution.

This solution was injected hypodermically at two positions in the backof 10-week male Balb/C mouse (from Japan SLC), and the blood was sampledat specific time intervals from the caudal vein. In the sampled blood,heparin of final concentration of 3.3 IU/ml was added, and plasma wascollected by centrifugal separation for 5 minutes at 5,000 rpm, and theconcentration of growth hormone in plasma was measured by the ELISAmethod.

By way of comparison, a non-granulated Exendin-4 solution (700 μg/0.2ml) was hypodermically administered in mouse, and the blood was sampledsimilarly.

To suppress antibody production by administration of Exendin-4, which isa dissimilar protein for mouse, three days before administration of theparticle, an immunosuppressant Tacrolimus hydrate (Astellas) washypodermically administered by 26 μg/mouse, and thereafter 13 μg/mousewas hypodermically administered at the time of the drug administration,and twice a week thereafter.

FIG. 21 shows time-course changes of Exendin-4 concentration in plasma.In the mouse administered non-granulated drug, the blood level in 1 hourafter administration was very high, and then dropped suddenly, to alevel before administration in a day. On the other hand, in the mouseadministered the microparticle drug prepared by using PEG-PLGA polymer,a transient elevation of blood level right after administration wassuppressed to about less than 1/100, and the blood level was sustainedat high levels for a month.

Example 31 Preparation of Microparticles Encapsulating FluoresceineLabeled Dextran (FD40) Different in Particle Diameter

5 mg of PEG-PLGA polymer (5k-55k) in Example 20 was dissolved in 100 μlof dimethyl carbonate to prepare a polymer solution of 50 mg/ml. In thispolymer solution, 20 μl of tert-butanol was added, and 20 μl of 1 mg/mlFD40 aqueous solution was dropped, and stirred by vortex to prepare areversed-phase emulsion. This reversed-phase emulsion was frozenpreliminarily by liquid nitrogen, and was freeze-dried by using afreeze-drying apparatus (EYELA, FREEZE DRYER FD-1000), at trap coolingtemperature of −45° C., and degree of vacuum of 20 Pa, for 24 hours. Theobtained solid content was dispersed in 50 μl, 200 μl, and 500 μl ofdimethyl carbonate to prepare an S/O suspension. This S/O suspension wasdropped in 2 ml of aqueous solution containing 10% Pluronic F-68 (aregistered trademark of BASF), and was stirred and emulsified in avortex mixer to prepare an S/O/W type emulsion. From this S/O/W typeemulsion, the water-immiscible organic solvent was removed by drying inliquid, and a microparticle dispersion liquid was obtained. Themicroparticle dispersion liquid was preliminarily frozen by liquidnitrogen, and was freeze-dried by using a freeze-drying apparatus(EYELA, FREEZE DRYER FD-1000), at trap cooling temperature of −45° C.,and degree of vacuum of 20 Pa, for 24 hours, and FD40-encapsulatingmicroparticle powder was obtained. The obtained microparticles wereobserved by a scanning electron microscope (SEM: HITACHI, S-4800), andthe average particle diameter was calculated.

FIG. 22 shows the correlation between the average particle diameter andthe amount of dimethyl carbonate added at the time of preparation ofS/O/W type emulsion. In a range from 50 μl to 500 μl, along withincrease of dimethyl carbonate amount, decline of the average particlediameter was observed.

INDUSTRIAL APPLICABILITY

A microparticle releases a hydrophilic active substance at anappropriate speed in the human body, and is useful as a DDSpharmaceutical preparation.

1. A microparticle, comprising an agglomerate of hydrophilic activesubstance containing particles, which particle comprises an amphiphilicpolymer composed of a hydrophobic segment of poly(hydroxy acid) and ahydrophilic segment of polysaccharide or polyethylene glycol, and ahydrophilic active substance.
 2. The microparticle according to claim 1,wherein the hydrophilic active substance containing particle has aninner hydrophilic segment of amphiphilic polymer, and an outer layer ofa hydrophobic segment of amphiphilic polymer.
 3. The microparticleaccording to claim 1, wherein the amphiphilic polymer is a gaftamphiphilic polymer composed of a polysaccharide main chain andpoly(hydroxy acid) graft chain(s).
 4. The microparticle according toclaim 3, wherein the polysaccharide main chain is dextran.
 5. Themicroparticle according to claim 1, wherein the amphiphilic polymer is ablock polymer composed of polyethylene glycol and poly(hydroxy acid). 6.The microparticle according to claim 5, wherein the average molecularweight of polyethylene glycol is 2,000 to 15,000.
 7. The microparticleaccording to claim 5, wherein a ratio of average molecular weight ofpoly(hydroxy acid) to average molecular weight of polyethylene glycol is4 or more.
 8. The microparticle according to claim 1, wherein thepoly(hydroxy acid) is poly(lactic acid-glycolic acid).
 9. Themicroparticle according to claim 1, wherein average particle diameter is1 to 50 μm.
 10. The microparticle according to claim 1, wherein thehydrophilic active substance is a peptide or a protein.
 11. A method formanufacturing a microparticle comprising: (a) forming a reversed-phaseemulsion by mixing an aqueous solvent containing a hydrophilic activesubstance and a water-immiscible organic solvent dissolving anamphiphilic polymer, (b) obtaining a solid content containing ahydrophilic active substance by removing the solvent from thereversed-phase emulsion, and (c) introducing the solid content or adispersion liquid containing the solid content into a liquid phasecontaining a surface modifier.
 12. The method according to claim 11,wherein the solvent is removed from the reversed-phase emulsion by afreeze-drying method.
 13. The method according to claim 11, wherein adispersion medium of the dispersion liquid containing the solid contentis a solvent capable of dissolving poly(hydroxy acid) and being 10 mg/mLor less in solubility of a hydrophilic segment for composing anamphiphilic polymer.
 14. The method according to claim 11, wherein theliquid phase is either an aqueous solvent or a water miscible organicsolvent.
 15. A pharmaceutical preparation comprising the microparticleof claim
 1. 16. The microparticle according to claim 2, wherein theamphiphilic polymer is a graft amphiphilic polymer composed of apolysaccharide main chain and poly(hydroxy acid) graft chain(s).
 17. Themicroparticle according to claim 2, wherein the amphiphilic polymer is ablock polymer composed of polyethylene glycol and poly(hydroxy acid).18. The microparticle according to claim 2, wherein a ratio of averagemolecular weight of poly(hydroxy acid) to average molecular weight ofpolyethylene glycol is 4 or more.
 19. The method according to claim 12,wherein a dispersion medium of the dispersion liquid containing thesolid content is a solvent capable, of dissolving poly(hydroxy acid) andbeing 10 mg/mL or less in solubility of a hydrophilic segment forcomposing an amphiphilic polymer.